Systems and methods for affixing seeds

Abstract

A receptacle for affixing a population of seeds includes a body having a top surface and bottom surface opposite the top surface. The body defines a well in fluid communication with the top surface. The well has a mouth disposed on the top surface and a tapered portion extending away from the mouth into the body and towards the bottom surface. The well is configured to receive and affix a seed of the population of seeds therein.

Description

SYSTEMS AND METHODS FOR AFFIXING SEEDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63662619, titled Systems and Methods for Affixing Seeds, filed on June 21, 2024, the contents of which are incorporated by reference herein in their entirety.TECHNICAL FIELD

[0002] Various embodiments of the present disclosure pertain generally to systems and methods for screening plant materials for certain attributes. More specifically, particular embodiments of the present disclosure relate to more rapid and efficient systems and methods for screening plant seeds, such as in breeding programs, by isolating DNA from the seeds while retaining seed viability. These methods and systems are useful for various plant seeds, such as crop seeds, grain seeds, vegetable seeds, and flower seeds.BACKGROUND

[0003] Plant research in the agriculture industry is performed to continuously provide genetic improvements in the germplasm, such as to introduce desired traits into plants (e.g., traits that enhance vigor, yield, disease resistance, drought resistance, herbicide tolerance, etc.). A number of different processes are used to introduce genetic improvements into plant germplasm, such as selective breeding, gene editing, genetic manipulations, targeted mutations, transformation, etc. Upon introduction of a desired genetic trait, plants are grown over multiple generations to ensure that the trait has become stably incorporated into the plant’s genome. However, due to differences in recombination, not all seeds of a plant will include the desired trait. Therefore, to develop a stable line, all seeds harvested from a plant typically need to be germinated and grown. Plants with the desired trait are moved forward in a breeding program while plants lacking or not expressing adequate levels of the desired trait are culled. In recent years, seed sampling methods have been developed wherein trait analysis is performed at the seed level to eliminate the need for germinating the seeds and growing the resulting plants, thereby reducing the greenhouse or field space requirement. Seeds with the desired trait are moved forward in the breeding pipeline while seeds not expressing adequate levels of the desired trait are culled.

[0004] One example approach for seed sampling is shown by Deppermann et al. in WIPO Publication No. W02006026466A2. Therein, each seed is “chipped” to remove a portion (or “chip”) that is analyzed for the presence of a desired trait. The seed is chipped such that the remainder of the seed retains viability and can be propagated, as needed.

[0005] However, the inventors herein have identified various issues with such approaches. In particular, there may be issues with sample handling. As one example, each seed has to be chipped with precision to ensure that embryonic matter is not removed, which could affect seed viability. Errors in sample removal can occur due to natural variations in seed size and shape To reduce errors, costly equipment may be required to visualize and orient the seed properly before it can be chipped. Also due to the precision with which only non-embryonic matter needs to be removed, the approach may be limited to certain types of seeds, such as those having a threshold size. For example, the approach may not be applicable to seeds that are too small to handle by the system (e.g., tomato seeds, lettuce seeds, flower seeds, cereal seeds, etc.). As another example, significant logistics are required to maintain the correct association of the seed chip (sample) with the corresponding chipped seed. In particular, since the sample is directed to a sample tray distinct from a seed tray where the chipped seed is directed, significant resources, such as laboratory information management systems / software (LIMS) are required to track the position of the sample in the sample tray and correlate it with the position of the seed in the seed tray, as well as to correlate the sample analysis data (e.g., DNA sequence data) with the identity of both the seed and the chipped sample. Typically, such LIMS systems can be cost, energy, and memory intensive. As still another example, due to the small size of the seed chip and its transportation along ducting via air pressure, there is a possibility of sample drift wherein the sample strays from the intended well of the sample tray to another well. This can corrupt sampling results, create an incorrect association between sample and seed, and cause other contamination issues. In other words, the approach requires precise seed holding, cutting, and manipulation of small seed chips and careful seed-to-chip identity tracking.

[0006] Another approach to seed sampling is disclosed by Hannappel et al. in U.S. Patent No. 10,011,828B2 wherein a maize seed is fully submerged in a solution from which DNA can be extracted. The seed is subsequently dried and germinated. However, the inventors herein have identified issues with this approach. As one example, the method requires a number of labware consumables. As another example, the approach may be limited by seed type. For example, submerging a soybean seed in an alkali solution may cause the seed coat to fall off and viability of the soaked seed to be compromised. As stillanother example, the alkali solution that is optimized for DNA extraction may not be compatible with downstream processes such as PCR or sequencing methods.

[0007] Accordingly, there is a need in the art for improved methods and systems for seed sampling while retaining seed viability. The present disclosure addresses at least some of the above issues by providing a method and system for in-seed sampling which simplifies sample collection and tracking, is compatible with a variety of seed types and downstream sample processing steps and maintains the viability of the sampled seed.SUMMARY

[0008] This disclosure relates generally to receptacles and methods for affixing a population of seeds for biological analysis. Specifically, the receptacles and methods described herein improve fixation of seeds during collection of biological material from seeds such as DNA.

[0009] In an aspect of the present disclosure, a receptacle for affixing a population of seeds includes a body having a top surface and a bottom surface opposite the top surface. The body defines a well in fluid communication with the top surface. The well has a mouth disposed on the top surface and a tapered portion comprising one or more walls extending away from the mouth into the body and towards the bottom surface. The well is configured to receive and affix a seed of the population of seeds therein.

[0010] In aspects, the one or more walls of the well are configured to exert a fixing force on the seed to hold the seed at a predetermined position and a known orientation within the well. The body may be configured to deform when the well receives the seed therein. The body may be configured to elastically deform in response to the seed being received within the well. The tapered portion may be configured to increase the fixing force exerted on the seed as elastic deformation of the body subsides. The tapered portion may slope inwardly away from the mouth at an angle between approximately 5 degrees and approximately 45 degrees from vertical. In particular embodiments, the one or more walls may comprise one single wall and the tapered portion of the well may have a frustoconical profile.

[0011] In aspects, the well includes a stem portion in fluid communication with the tapered portion. The stem portion may extend from an apex of the well towards the bottom surface of the body. The apex may be spaced apart from the mouth by the tapered portion. The stem portion may have a cylindrical profile. The tapered portion may have a first diameter and a second diameter. The first diameter of the tapered portion may be equal to adiameter of the mouth and the second diameter of the tapered portion may be equal to a diameter of the stem portion. The stem portion may be in fluid communication with the bottom surface such that the well extends entirely through the body and the top surface and the bottom surface are in fluid communication therethrough.

[0012] In particular aspects, the tapered portion has a circular cross-section. The tapered portion may alternatively have an oblong cross-section. The mouth may be spaced apart from the top surface such that the well defines a head space between the top surface and the mouth.

[0013] In some aspects, the receptacle has a plurality of other wells forming an array of wells. Each well of the array of wells may be configured to receive and affix a respective seed of the population of seeds. The array of wells may be distributed uniformly across the top surface of the body.

[0014] In certain aspects, the body is made of a deformable material. In particular embodiments, the deformable material may be a polyisocyanurate foam or a phenolic foam. The foam material may have a density of approximately 2 lbs / ft3. The body may be formed as a monolithic construction.

[0015] In particular aspects, a system for affixing a population of seeds includes a receptacle and an aligner plate. The aligner plate may be configured to position the seed within a well of the receptacle at a known depth.

[0016] In another aspect of the present disclosure, an apparatus for affixing a population of seeds includes a receptacle and an aligner plate. The receptacle has a top surface and bottom surface opposite the top surface. The receptacle defines a well in fluid communication with the top surface. The well has a mouth disposed on the top surface and a tapered portion comprising one or more walls extending away from the mouth into the receptacle and towards the bottom surface. The well is configured to receive a seed of the population of seeds to hold and affix the seed therein. The aligner plate is configured to position the seed within the well. The aligner plate includes a prong that positions the seed within the well at a known depth. The seed elastically deforms the receptacle when the seed is positioned such that the well exerts a fixing force on the seed as elastic deformation of the receptacle subsides.

[0017] In aspects, the well defined by the receptacle includes a tapered portion sloping inwardly from the top surface at an angle between 5 degrees and 80 degrees from vertical. The tapered portion may be configured to increase the fixing force exerted by the wall of the well on the seed to hold the seed at a predetermined position and a knownorientation within the well. The tapered portion may be configured such that the fixing force increases as elastic deformation of the receptacle subsides.

[0018] In some aspects, the aligner plate deforms the receptacle about the well such that the well includes a tapered portion. The tapered portion may be formed in situ when the aligner plate positions the seed within the well. The aligner plate may deform the receptacle such that the tapered portion extends away from the top surface towards the bottom surface. The tapered portion may slope inwardly at an angle between 5 degrees and 45 degrees from a vertical reference axis.

[0019] In particular aspects, the receptacle includes a plurality of other wells forming an array of wells. Each well of the array of wells may be configured to receive a respective seed of the population of seeds to hold and affix each seed. The aligner plate may be configured to position each seed of the population of seeds at a known depth simultaneously. The aligner plate may include a plurality of prongs forming an array of prongs. Each prong of the array of prongs may be configured to position a respective seed within a respective well such that each seed elastically deforms the receptacle when the aligner plate positions each seed within a respective receptacle. Each prong of the array of prongs may be configured to deform the receptacle such that each well of the array of wells includes a tapered portion when the aligner plate positions each seed within a respective well. Each tapered portion may be formed in situ when the aligner plate positions the population of seeds within the wells. The aligner plate may be configured to position each seed of the population of seeds at the same depth within a respective well relative to the other seeds of the population of seeds. The aligner plate may be configured to position each seed of the population of seeds at the same depth within a respective well such that the crown of each seed is coplanar with the crown of other seeds of the population of seeds affixed in the receptacle.

[0020] In another aspect of the present disclosure, a method of affixing a population of seeds includes inserting a seed of the population of seeds into a well of a receptacle, the well having a tapered portion comprising one or more walls, and positioning the seed at a known depth within the well such that the tapered portion exerts a fixing force on the seed.

[0021] In aspects, the method includes allowing the seed to remain in the receptacle undisturbed for a rest period prior to subjecting the seed to an analysis process. Positioning the seed may include using an aligner plate to dispose the seed at the known depth. The aligner plate may elastically deform the tapered portion contemporaneously when positioning the seed.

[0022] In some aspects, positioning the seed includes disposing the seed at a depth equal to a depth of another seed of the population of seeds inserted into another well of the receptacle such that the crown of each seed is coplanar with one another. In some embodiments, inserting the seed may include orienting the seed with respect to the well. Inserting the seed in the well may be performed manually or may be automated.

[0023] It is to be understood that both the foregoing general description and the following detailed description are examples and explanatory only and are not restrictive of the disclosed embodiments, as claimed.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various example embodiments and together with this description, serve to explain the principles of the disclosed embodiments.

[0025] FIG. 1 is a schematic depiction of a method for in-seed sampling in accordance with embodiments of the present disclosure applicable to a population of seeds;

[0026] FIG. 2 is a high-level flowchart illustrating the method of FIG. 1;

[0027] FIG. 3 is a perspective view of a receptacle for receiving and affixing seeds for in-seed sampling in accordance with embodiments of the present disclosure;

[0028] FIG. 4 is a cross-sectional view of the receptacle taken along section line A- A of FIG. 3;

[0029] FIG. 5 is a cross-sectional view of another receptacle taken along section line A-A of FIG. 3;

[0030] FIG. 6 is a cutaway view of another receptacle in accordance with embodiments of the present disclosure;

[0031] FIG. 7 is a cross-sectional view of another receptacle taken along section line A-A of FIG. 3;

[0032] FIG. 8 is top elevation view of another receptacle in accordance with embodiments of the present disclosure;

[0033] FIG. 9 is a perspective view of an aligner plate for use with a receptacle in accordance with embodiments of the present disclosure;

[0034] FIG. 10 is a perspective view of another aligner plate;

[0035] FIG. 11 is an example of the aligner plate of FIG. 10 in use with a receptacle for aligning seeds in the receptacle to a common depth or plane;

[0036] FIG. 12 is an example well tool for forming a well of a receptacle in use prior to forming the well;

[0037] FIG. 13 is the well tool of FIG 12 in use during formation of the well of a receptacle;

[0038] FIG. 14 is a perspective view of a press in accordance with embodiments of the present disclosure;

[0039] FIG. 15 is a flowchart of a method for affixing a seed in a receptacle in accordance with embodiments of the present disclosure;

[0040] FIG. 16 is an example embodiment of a drilling device that may be used to create an orifice for exposing a portion of a seed or population of seeds for analysis;

[0041] FIG. 17 is perspective view of the receptacle of FIG. 3 with segments of a sacrificial portion thereof utilized;

[0042] FIG. 18 is side elevation view the receptacle of FIG. 17;

[0043] FIG. 19 is an example embodiment of a cleaning tool that may be used to remove debris from the orifice of a seed following, or concurrent to, the action of an associated drilling device;

[0044] FIG. 20 is a flowchart of another method for in-seed sampling in accordance with embodiments of the present disclosure;

[0045] FIG. 21 is a real-time amplification plot depicting relative DNA yield and quality from an in-seed sampling test in accordance with the disclosed embodiments;

[0046] FIGS. 22 and 23 are allelic PCR endpoint plots depicting allelic separation in DNA isolated using from an in-seed sampling test of corn seed in accordance with the disclosed embodiments; and

[0047] FIG. 24 is an allelic PCR endpoint plot depicting allelic separation in DNA isolated using from an in-seed sampling test of soy seed in accordance with the disclosed embodiments.DETAILED DESCRIPTION

[0048] Reference will now be made in detail to the example embodiments of the present disclosure, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.

[0049] The systems, devices, and methods disclosed herein are described in detail by way of examples and with reference to the figures. The examples discussed herein areexamples only and are provided to assist in the explanation of the apparatuses, devices, systems, and methods described herein. None of the features or components shown in the drawings or discussed below should be taken as mandatory for any specific implementation of any of these devices, system, or methods unless specifically designated as mandatory. Features from one embodiment or aspect can be combined with features from any other embodiment or aspect in any appropriate combination. For example, any individual or collective features of method aspects or embodiments can be applied to apparatus, product, or component aspects or embodiments and vice versa.

[0050] Also, for any methods described, regardless of whether the method is described in conjunction with a flow diagram, it should be understood that unless otherwise specified or required by context, any explicit or implicit ordering of steps performed in the execution of a method does not imply that those steps must be performed in the order presented but instead may be performed in a different order or in parallel. As used herein, the term "exemplary” is used in the sense of “example," rather than “ideal.” Moreover, the terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of one or more of the referenced items.

[0051] The present disclosure provides a method for in-seed sampling comprising holding and fixturing a seed, removing a region of the seed to expose an area of endosperm or cotyledon (or other portion of the seed), disposing the removed region so that a “sampling region” is created in situ in the seed, pipetting a solution for analysis directly into contact with the exposed sampling region of the seed, and based on the analysis, determining whether to select the seed for progress into a breeding pipeline. A detailed description of the in-seed sampling systems and methods contemplated herein are further described in International Patent Application Serial No. PCT / US23 / 85005, the systems and methods disclosed therein being hereby incorporated by reference. In some embodiments, at least some of the steps of the method are automated and do not require user input, while other steps may require user input. In other embodiments, all the steps of the method are automated and do not require user input. The disclosure also includes apparatuses, individual components, and systems for performing the in-seed sampling methods as described herein. In certain embodiments, the apparatuses, individual components, and systems are semi-automated (i.e., requiring some manual input) or fully automated. Further, the disclosure includes methods and systems, including automated systems and methods for liquid DNA, protein, or RNA isolation, seed storage, as well as seed selection and retrieval. It should be noted that in some embodiments,a portion of the seed other than the cotyledon or endosperm may be exposed for subsequent seed analysis, such as, for example, the seed coat.

[0052] As elaborated herein, the methods and systems of in-seed sampling provide various advantages. First, by removing a portion of the seed to create an orifice in the seed and disposing of the removed seed debris, the seed effectively acts as both the seed and the sample. By eliminating the need for a sample that is distinct from the seed, a variety of sample handling issues such as sample drift and the need for complex sample tracking systems (e g., expensive LIMS systems) is reduced. Further, the requirement for only a portion of the seed to be exposed for in-seed sampling allows for customization of a cutting device (e.g., a drill) and a cutting operation (e.g., drilling operation) based on seed type. For example, different cutting tools can be developed based on seed size and shape, seed coat thickness, seed oil content, and similarly cutting operations may extend the cutting device to different depths of the seed based on the relative position of the embryo in the seed relative to the tissue, e.g., the endosperm or cotyledon. Further, although in some embodiments the dispensed solution may come in contact with at least a portion of the seed coat, in other embodiments, contact between the dispensed solution and the seed coat may be purposely minimized. For example, in some embodiments, a combination of the volume of the in-seed orifice created by the cutting device and the components of the solution dispensed into the seed for extracting a biological molecule therefrom (e.g., solutions for extracting DNA, RNA, protein, carbohydrate, etc.) may be optimized. As such, exposure of the seed contents to the solution may be limited, which may improve seed viability, and germination efficiency and reduce seed coat fall-off issues.

[0053] The embodiments described in the present disclosure also allow for usage of fewer consumables, such as fewer seed trays and sample trays, labware, and reagents by virtue of making the seed itself a vessel for the liquid handling step. By eliminating the need for distinct sample trays and seeds trays, the need for complex tracking systems for correlating the trays is also reduced. In some embodiments, the receptacle into which seeds are affixed for performing the in-seed sampling may be reusable. Depending on the extraction protocol used for the biological molecule(s), this approach can also eliminate the need for lab materials related to shaking, grinding, centrifugation, and incubation. Overall, a rapid in-seed sampling method and system can be enabled with fewer steps and components.

[0054] Selective seed breeding has been made more systematic by procedures that allow the plant breeder to isolate DNA material. DNA analysis allows the breeder to identify seeds with preferred genetic characteristics, and to use only those seeds to propagate progenyplants from which additional seeds are selected to be harvested. Particular embodiments of the present disclosure enable in-seed DNA sampling by cutting into (e.g., drilling) and exposing a portion of the inner seed region following by direct application of a solution, configured for extracting DNA, to the exposed inner seed region. The methods result in an in-seed solution comprising seed DNA, and a viable seed. The in-seed sampling method can be similarly used to extract and analyze proteins, carbohydrates, RNA, and / or other biological molecules from a seed. By analyzing the biological molecule (e.g., DNA) from the in-seed solution, a breeder can quickly determine if the seed should be propagated or culled. By enabling earlier determination of seed characteristics, greenhouse and field resource usage can be minimized and seed selection decisions can be made earlier in the breeding pipeline, if desired. For example, seeds can be advanced into a Single Seed Descent (SSD) program in an earlier generation (than otherwise possible), if desired.

[0055] The disclosed methods, apparatuses, and systems are also compatible with existing and upcoming DNA sequencing-based technologies, such as Skim sequencing and Genotyping by Sequencing (GBS), including target Genotyping by Sequencing (tGBS), due to the need for smaller DNA liquid volumes (about 4-10 L, depending on pre-QC steps, for multiple assays). The methods and apparatus and systems are also compatible with legacy genotyping assays like Taqman or KASP, where higher volumes of DNA and / or DNA concentration can be required. In systems that require larger volumes, including legacy genotyping chemistries, the in-seed extraction step can be repeated multiple times, or an additional cavity can be formed in the seed to hold a larger volume. Furthermore, the extraction solution can be configured to be compatible with the downstream PCR and sequencing approaches.

[0056] An example method for in-seed sampling is now discussed with reference to FIG. 1 (which shows a schematic representation of an example method 100) and FIG. 2 (which shows a high-level flowchart 200 of the example method 100). For example, the steps described in the flowchart 200 may correspond to the steps of the method 100 shown schematically with the leading “1” of a respective label replaced with a leading “2”). Some embodiments of the method of FIGS. 1 and 2 may be performed by an in-seed sampling device or system automatically and / or autonomously, without user input, such as by a robot optionally aided by machine vision. In other embodiments, the method may be performed semi-automatically wherein at least some of the steps are performed automatically, e.g., by a robot, while at least some other steps are performed manually by a user. One or more example embodiments of components, device, tools and / or apparatus that can be used toperform one or more steps of the disclosed method are introduced at FIGS. 1 and 2 and elaborated in further detail thereafter.

[0057] The in-seed sampling method 100, 200 (FIGS. 1 and 2, respectively) starts with obtaining a seed, or a population of seeds (step 10 of FIG. 1; step 20 of Fig. 2). In example embodiments, obtaining a population of seeds 10, 20 may comprise obtaining a collection of seeds for sampling from a container, such as, for example, seeds contained in a seed packet or seed bag. In some examples, an identifier on the bag (e.g., barcode or other “bag tag”) may be scanned and an identity of the seed packet or seed bag may be saved in a data management system associated with the in-seed sampling device. In some embodiments, the population of seeds 10 is a bulked population or a single seed descent population. In other embodiments, the population of seeds 10 includes hybrid seeds from a selected parent plant (e.g., all F2 or all F3 seeds from a plant, plant line, or plant crossing) or inbred or backcrossed seeds (e.g., BC1, BC2, BCn, etc.). The seed(s) 10 may be sourced from any plant. As non-limiting examples, the seed(s) 10 may be a crop seed (e.g., seeds from com (dent / field com or sweet corn), soy, sunflower, rapeseed, cotton, etc.), cereal seeds (e.g., seeds from corn, barley, wheat, rice, oat, etc.), vegetable seeds (e.g., seeds from bean, brassica, cucumber, lettuce, okra, pepper, spinach, squash, tomato, etc.), fruit seeds (e.g. seeds from melons including watermelon, pear, apple etc.), flower seeds, as well as tree seeds. For purposes of the present disclosure, and the following descriptions, a population of seeds 10 may comprise a single seed 10 or multiple seeds 10 (e.g., more than one seed such as a bulk population of seeds).

[0058] After obtaining a seed or population of seeds 10, the method includes affixing the seed or a population of seeds into a receptacle (Step 101, 201). An example embodiment of such a receptacle 300 is provided at FIG. 3. In some embodiments, affixing the seed 10 includes orienting the seed 10 in a predefined orientation (e.g., placing the seed in a predetermined position that facilitates subsequent seed sampling). As such, each seed of the population of seeds is affixed into the receptacle in a common, predefined orientation. In other embodiments, such as where the seed 10 is self-orienting, affixing the seed includes placing the seed(s) 10 in the receptacle 300 without the need for active orientation adjustments. For example, the seed(s) 10 may be dispensed into the receptacle 300 from a hopper while shaking the receptacle 300 so that the seeds 10 are oriented (e.g., self-oriented) upon entering the receptacle 300. Additionally, or alternatively, the ducting via which the seeds 10 are released from the hopper into the receptacle 300 may be shaken.

[0059] Still other known methods of seed singulation may be used to dispense seeds into the receptacle, such as those described with reference to seed loading in US9, 551,636 at Figure 3, the disclosure of which is incorporated by reference herein.

[0060] In some embodiments, the common, predefined orientation in which the seeds 10 are affixed in the receptacle 300 may include each seed 10 being coplanar or tangential with a surface of the receptacle 300, so that the exposed side or area of each seed 10 of the population of seeds 10 is uniform. In one embodiment, a crown of each seed 10 is used to position the population of seeds 10 in the common orientation. For example, the crown may be aligned such that the crown is coplanar with or tangential to a top surface 304 of the receptacle 300. In embodiments, individual wells 310 of the receptacle 300 may be sized and shaped to receive a corn seed / kemel in the predefined common orientation where the bottom of the com kernel extends into a well 310 while the crown of the com kernel extends out of the well 310 or is approximately coplanar with the top surface 304 of the receptacle 300. In other embodiments, the well 310 of the receptacle 300 may be configured to receive the seed 10 in any orientation and upon shaking the receptacle 300, the seeds 10 may self-orient. This may be advantageous for self-orienting seeds such as soy seeds. In still further embodiments, the wells 310 of the receptacle 300 may be configured to receive the seeds 10 in a flat orientation with the seed 10 lying on a dorsal or ventral side. This may be advantageous for seeds that are less rounded, such as pumpkin seeds or sunflower seeds.

[0061] With particular reference to FIG. 3, a receptacle 300 used for affixing the seeds 10 for in-seed sampling in accordance with embodiments of the present disclosure is shown. The receptacle 300 includes a body 302 having a top surface 304 and a bottom surface 306. The body 302 defines a plurality of wells 310 to receive the seeds 10 for sampling or analysis. The plurality of wells 310 may be arranged in an array. The array may follow microplate standards established by the Society of Biomolecular Screening (SBS). For example, the array may have ninety-six (96) wells 310 arranged in an 8x12 format with a 9mm pitch on both columns and rows. The array of wells 310 may be distributed uniformly over the top surface 304. In embodiments, the body 302 may have an outer footprint of approximately 127mm x 85 mm. In some embodiments, the array may have 8, 12, 24, 48, 96, 384, 1536 wells, or any other array of wells 310 routinely used with standard automated liquid handling platforms. Conforming to a standard established by the SBS allows for compatibility of the receptacle 300 with a wide variety of off-the-shelf liquid handlers, plate storage robots, robotic plate stackers, plate moving robots, plate moving axis / axes, and analysis tools for laboratory automation. In some embodiments, the receptacle 300 is labeledwith an identifier 303, such as with a barcode, QR code, RFID, NFC, Bluetooth, UWB, and / or pick-to-light tracker or other human-readable or machine-readable identifier, to allow for ease of tracking. Further still, the top surface 304 of the receptacle may have identifiers 303 or markings identifying each well 310 of the receptacle 300.

[0062] The receptacle 300 applies a fixing force to the seeds 10 received within the wells 310 to hold and stabilized the seeds 10 in the desired orientation and position. The fixing force may be a positive force, e.g., a compressive force, a friction force, or a combination of a positive force or friction force. The receptacle 300 may be made of a deformable material. For example, the receptacle 300 may be made of a foam material. The foam material may be an open cell foam or a closed cell foam. The material of the receptacle 300 may exhibit both plastic deformation and elastic deformation characteristics. As used herein, the term “plastic deformation” refers to permanent or non-rever sable deformation in response to applied forces and the term “elastic deformation” refers to temporary or reversable deformation in response to applied forces. In particular embodiments, the receptable is made of a material that exhibits higher plastic deformation than elastic deformation, e.g., less than full or complete plastic deformation. As a result of the elastic deformation of the receptacle material caused by positioning the seed 10 within the well 310, the receptacle 300 may apply the fixing force on the seed 10 within the well 310. For example, positioning the seed 10 within the well 310 may deform a portion of the well 310. The well 310 may return or partially return to its original dimension due to the elastic deformation of the body 302. When the well 310 returns to its original dimension, walls of the well 310 may exert a clamping force or fixing force on the seed 10 within the well 310. In particular embodiments, the foam material is a polyisocyanurate foam, e.g., ISO 2000 foam, ISO 3500 foam, ISO 4000 foam, or ISO 6000 foam produced by ISO Technologies, Inc. 200 Milliken Dr., Hebron, OH 43025. The foam may have a density in the range of 0.5 Ibs. / ft3to 5 Ibs. / ft3, e.g., 2 lbs. / ft3. In particular embodiments, the receptacle 300 is formed or molded as a monolithic construction.

[0063] In other embodiments, the receptacle 300 may be made of a nondeformable material, e g., a metal material, or a rigid plastic material. In certain embodiments, the receptacle 300 is made of natural deformable material or biodegradable material, e.g., a wood-based material, corn or soy or bamboo-based plastic material, corrugated paper, or cardboard, etc. In some embodiments, the receptacle 300 is made of inexpensive plastic or foam so that the process can be inexpensively scaled up to use a plurality of receptacles 300. In still further embodiments, the receptacles 300 may be configured to be reused. In exampleembodiments of a reusable receptacle, a second array of wells 310 are disposed on the bottom surface 306 of the receptacle 300, the second array of wells 310 aligned with, or offset from, the first array Upon use of the first array for sampling a first set of seeds, the first array may be disposed or retained and the second array may be used or sampling a second set of seeds.

[0064] With additional reference to FIGS. 4 and 5, each of the wells 310 has a funnel or a tapered portion 312. The tapered portion 312 is responsible for the increased fixing force exerted on a seed 10 received within the well 310. Additionally or alternatively, the tapered portion 312 may accommodate various sized seeds having an irregular shape. In embodiments, the tapered portion 312 may receive a seed 10 such that the seed 10 self-orients within the well 310. In certain embodiments, the tapered portion 312 may provide a clearance between the seed 10 and the body 302 of the receptacle 300 to ease sampling of the seed within a well 310. For example, the tapered portion 312 may provide additional clearance for a cutting device (for example, the cutting device 600 of FIG. 16) for cutting an orifice in the affixed seed 10. The tapered portion 312 extends away from the top surface 304 of the receptacle into the body 302 and towards the bottom surface 306. The well 310 has a mouth 314 and an apex 316. The mouth 314 is the part of the well 310 positioned nearest the top surface 304, immediately adjacent to the tapered portion 312, while the apex 316 is the part of the well 310 positioned farthest from the top surface 304, immediately adjacent to the tapered portion 312. The tapered portion 312 of the well 310 is defined such that the well 310 tapers inwardly from the mouth 314 to the apex 316. The tapered portion 312 may taper at a slope 0 that is less than 90 degrees from a vertical axis (indicated by dashed lines). For example, the slope 0 may be in the range of 5 degrees to 80 degrees, e.g., 8, 10, 12, 15, 20, 25, 35, 50, or 75 degrees from the vertical axis. The mouth 314 of the well 310 may be disposed on the top surface 304. In various embodiments, the tapered portion 312 may comprise a single wall or more than one wall. In embodiments where the tapered portion 312 has a single wall, the tapered portion 312 and / or the well 310 may have a circular crosssection, as shown in FIG. 3. In other embodiments, the tapered portion 312 may have three walls and may have a triangular profile. In some embodiments, the tapered portion 312 may have four walls and may have a square or rectangular cross-section. In certain embodiments, the tapered portion 312 may have a larger number of walls such as five, six, seven, eight, or more than eight walls. The profile of the tapered portion 312 and / or the well 310 is defined by the number of walls of the tapered portion 312. For example, where the tapered portion 312 has a single wall, the tapered portion 312 may have a frustoconical profile with the apex 316 having a diameter less than a diameter of the mouth 314. In example embodiments, themouth 314 may have a diameter in the range of 0.2 millimeters to 20 millimeters, e.g., 2, 4, 6, 8, or 10 millimeters. The apex 316 may have a diameter in the range 0.1 millimeter to 20 millimeters, e g., 1, 2, 3, 4, 5, 6, or 8 millimeters. In some embodiments where the tapered portion 312 has a single wall, the tapered portion 312 has a conical profile with the apex 316 terminating in a vertex, e.g., at a sharp point. For example, a tapered portion 312 having a conical profile may be suited for affixing narrow seeds 10. In embodiments, where the tapered portion 312 has three or four walls, the tapered portion 312 may have a frustopyramidal or pyramidal profile. The walls of the tapered portion 312 may be any combination of straight and curved walls. For example, the tapered portion 312 may have a stadium cross-section with two opposing flat walls and two opposing curved walls. The different dimensioned diameters of the mouth 314 or the apex 316 may be provided to accommodate different seed types and girths, e.g., corn, soybeans, pumpkin seeds, sunflower seeds, tomato seeds, or other seeds.

[0065] In certain embodiments, the wells 310 have a stem portion 318. The stem portion 318 may receive a seed 10 or a portion of a seed 10. The stem portion 318 extends from the apex 316 of the tapered portion 312 toward the bottom surface 306. The stem portion 318 may terminate short of the bottom surface 306 such that the well 310a is a blind hole (FIG. 4) or may extend through to the bottom surface 306 such that the well 310b is a through hole (FIG. 5). The stem portion 318 may have a constant or uniform diameter such that the stem portion 318 has a cylindrical profile. The stem portion 318 can intersect the apex 316 of the tapered portion 312 such that the tapered portion 312 has a frustoconical profile as shown in FIGS. 4 and 5. The diameter of the stem portion 318 may be equal to the diameter of the apex 316. In embodiments, as shown in FIG. 4, the stem portion 318 of each well 310 of the plurality of wells 310 may extend into the body 302 so as to be at a uniform depth. In some embodiments, the stem portion 318 of the plurality of wells 310 may extend into the body 302 such that the wells 310 have varying depths as shown in FIG. 6. The array of wells 310 may be organized such that each well 310 in a respective row or a respective column of the array has the same depth. For example, a first row of the array may have wells 310 with a stem portion 318a extending to a first depth DI into the body 302, a second row of the array may have wells 310 with a stem portion 318b extending a second depth D2 into the body 302, and a third row of the array may have wells 310 with a stem portion 318c extending a third depth D3 into the body 302.

[0066] Referring to FIG. 7, in embodiments, the receptacle 300 may define a well 310c. The well 310c is defined such that the mouth 314 and the tapered portion 312 arepositioned below or spaced apart from the top surface 304. A receptacle 300 having an array of wells 310c may be advantageous for affixing certain seed types, e.g., soy seeds. In such embodiments, the well 310 is defined to include a head space 311 between the top surface 304 and the mouth 314. The head space 311 may accommodate an additional volume of soak solution. In embodiments, the receptacle 300 may include any combination or mixture of wells 310a, 310b, or 310c. For example, the receptacle 300 may include wells 310a and wells 310b, or wells 310a and wells 310c, or wells 310b and wells 310c, or each of wells 310a, 310b, and 310c.

[0067] Referring to FIG. 8, in embodiments, the receptacle 300 may define a well 3 lOd. The well 3 lOd is defined to have an oval or oblong or elliptical shape, as opposed to a circular shape. For example, the well 3 lOd may be considered to have a stadium crosssection with two opposing flat walls and two opposing curved walls. In embodiments, both the tapered portion 312 and the stem portion 318 of the well 3 lOd are oblong. In some embodiments, only the tapered portion 312 of the well 310d is oblong, while the stem portion 318 is circular. In certain embodiments, the tapered portion 312 of the well 310d is circular while the stem portion 318 is oblong. A receptacle 300 having an array of wells 3 lOd may be advantageous for affixing certain seed types that are relatively thicker along their length compared to their width or depth, e.g., corn kernels which could range from 3mm up to 18mm on their longest dimension. In embodiments, the receptacle 300 may include any combination or mixture of wells 310a, 310b, 310c, and / or 3 lOd.

[0068] In particular embodiments, the tapered portion 312 of a well 310 may function as an ex-seed soak containment region in the well 310 of the receptacle 300 that is fluidly coupled to the in-seed soaking region after an orifice is created in the affixed seed 10 during step 104, 204 of method 100, 200 detailed below. The ex-seed soaking region may facilitate seed preprocessing and / or handling of larger soak solution volumes. For example, after affixing the seeds 10, a preprocessing solution may be delivered to the ex-seed soaking region to pre-soften at least the crown of the seed 10 thereby improving the efficiency of creating an orifice in the seed crown (e.g., by requiring less force or less duration) and / or improving yield of biological materials (e.g., DNA) from the seed 10 during sampling. In various embodiments, the amount of solution that can be contained in the ex-seed containment region in the well, above the affixed seed, during step 104, 204 of methods 100, 200 may vary. For example, the volume of the ex-seed containment region may be 5pL, 50|iL, lOOpL, lOOOpL, 2000pL, or more than 2000pL. Furthermore, in various embodiments the extent to which the seed coat is in contact with the solution may vary. The amount of theseed coat in contact with the solution may be controlled by the volume defined by tapered portion 312 of the wells 310 or by the position of the seed 10 within the well 310. In certain embodiments less than 75% of the seed coat surface area is in contact with the solution, such as less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, and or less than 1% of the seed coat surface area may be in contact with the solution. In other embodiments include, the percentage of the overall seed coat surface area that is in contact with the solution in the ex-seed containment region is 1 - 5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-40%, or more, of the seed coat area, or any value therebetween.

[0069] In some embodiments, the receptacle 300 includes a separate lid. The lid may prevent contamination of seeds held in the receptacle 300. In some embodiments, the lid may also prevent or reduce evaporation of a solution applied to the seeds 10.

[0070] Referring now to FIG. 9, an example embodiment of an aligner plate 500 that may be used to position the seeds 10 to a common depth or plane in the receptacle 300 is shown. The aligner plate 500 includes a number of prongs 501 (e.g., elongate pushers or extensions) that correspond to the positions of actual or desired wells 310 in the receptacle 300 for affixing the seeds 10. Although in the embodiment depicted in FIG. 9, the prongs 501 are substantially cylindrical and substantially the same length, in other embodiments the prongs may have different shapes and / or may have varying lengths so as to position the seeds 10 to varying depths (e.g., with reference to the receptacle of FIG. 6). The prongs 501 may have a diameter between 1 and 20 millimeters, such as between 3 millimeters to 15 millimeters. In embodiments, the diameter of the prongs 501 is less than a statistically significant value (e.g., mean, mode, or median value) of the diameter of population of seeds 10, or less than the diameter of the smallest seed 10 in the population of seeds. When the diameter of the prong 501 is less than the diameter of the seeds, the likelihood of seeds rotating or twisting within the well, during positioning or affixing within the well 310, is reduced.

[0071] Referring to FIGS. 10 and 11, another aligner plate 550 in accordance with embodiments of the present disclosure is shown. The aligner plate 550 may be used to position the seeds 10 in the wells 310 of the receptacle 300. The aligner plate 550 includes a number of prongs 552 corresponding to the number and the position of the wells 310. The prongs 552 include a tapered segment 554. The tapered segment 554 is received in the tapered portion 312 of a respective well 310 when the aligner plate 550 positions the seeds 10 within the tapered portion 312 of the wells 310. The aligner plate 550 may include legs 558to align the prongs 552 with respect to the receptacles 300. The legs 558 are positioned and spaced to receive the body 302 therebetween. The legs 558 may engage the body 302 to align the prongs 552 relative to the receptacles 300.

[0072] Referring to FIGS. 12 and 13, in particular embodiments, a well tool 800 may be used to form the wells 310 in the receptacle at a time of performing the method 10, 20, and before affixing the seeds in the wells. In embodiments, the well tool 800 may form the tapered portion 312 and / or the stem portion 318 of the receptacle 300 upon coupling with the receptacle. The body 302 of the receptacle 300 may be prescored and / or drilled to define dimples 305 or divots or other markings to indicate desired sites of seed affixation so that an array of the affixed seeds 10 can be easily formed in the receptacle 300 upon use of the well tool. For example, the well tool 800 may be plunged or driven into a solid block of material to form the wells 310 of the receptacle 300 before the seeds are affixed therein. The well tool 800 may be configured to form the wells 310a, 310b, 310c, 310d substantially instantly as described above. The well tool 800 may be used manually or may be used in an automated process.

[0073] The well tool 800 includes tines 802 that form each well 310. Each tine 802 includes a tapered section 804 and may include a post section 806. The tapered section 804 forms the tapered portion 312 of a respective well 310. The post section 806 extends from the tapered section 804. In embodiments including the post section 806, the post section 806 forms the stem portion 318 of a respective well 310. The post section 806 can extend from the tapered section 804 at various lengths such that the stem portion 318 is formed with various lengths. For example, the well tool 800 may include tines 802 having a post section 806a with a first length, tines 802 having a post section 806b with a second length, or tines 802 having a post section 806c with a third length, or no post section 806. The post section 806 may have a profile that matches the profile of the stem portion 318, e.g., a circular crosssection or a cylindrical profile. The well tool 800 may be similar to the aligner plate 500, 550 in size and dimension. The well tool 800 may be distinguished from the aligner plate 500, 550 by the inclusion of the post section 806 or a tapered section 804 being of larger dimension, e.g., longer, than the tapered segment 554 of the aligner plate 550 or of a larger diameter.

[0074] Referring to FIG. 14, a press 570 in accordance with embodiments of the present disclosure is shown. The press 570 holds the receptacle 300 and the aligner plate 500, 550 for positioning the seeds 10 within the receptacle 300. In some embodiments, the press holds the well tool 800 for forming the wells 310 in the receptacle 300. The press 570includes a base 572 and a plunger 574. The base 572 receives the receptacle 300. The base 572 may have a fixture arm 576 to locate and fix the receptacle 300 with respect to the plunger 574. In embodiments, the fixture arm 576 may be configured as spring-loaded lever arms or spring-loaded locating pins. The aligner plate 500, 550 (or the well tool 800) can be secured to the plunger 574. The plunger 574 is depressed or “plunged” towards the base 572 to position the seeds 10 within the well 310 or to form the wells 310.

[0075] Returning to FIGS. 1-2, in the depicted embodiment the seeds 10 are affixed into the receptacle 300 manually (e.g., by an operator), or through the use of automation (e.g., using a multi -axial robot arm). Manually affixing the seeds 10 into the receptacle 300 may include a user placing the seeds 10 into wells 310 in a desired orientation and using the aligner plate 500, 550 to position the crown of the seeds 10 coplanar with each other. The desired orientation is a position in which the seeds are held in the receptacle that facilitates subsequent seed sampling. In some embodiments, the desired orientation is one where a region of the endosperm or cotyledon is exposed for subsequent cutting (e g., drilling) steps 102, 202 described below. As noted above, in some embodiments where the receptacle 300 is made of a material that is deformable e.g., phenolic or polyisocyanurate foam material, manually affixing the seeds 10 may include the user pushing the seeds 10 into the receptacle material to instantaneously form the wells 310 wherein the seed 10 is embedded in the desired orientation. The material of the body 302 may be prescored and / or drilled (e.g., with dimples or other markings) to indicate desired sites of seed affixation so that an array of the affixed seeds 10 can be easily formed in the receptacle 300. In one example, the receptacle 300 is prescored so that seeds 10 can be affixed in a 1, 2, 4, 8,12, 24, 48, 96, 384, or 1536 well configuration.

[0076] With additional reference to FIG. 15, a method 1200 of affixing the seed 10 or a population of the seeds 10 for sampling and analysis in accordance with embodiments of the present disclosure is described with reference to the receptacle 300, the aligner plate 500, 550, the well tool 800, and the press 570 of FIGS. 3-14. The method 1200 may be considered a subroutine of steps 101, 201 of methods 100, 200. A receptacle 300 may be received on a worksurface for sampling seeds (step 1210). The work surface may be a table or the surface 602 of a cutting device 600 (FIG. 16) or the base 572 of the press 570. In embodiments, the receptacle 300 is received on the work surface with the wells 310 already formed. Alternatively, in certain embodiments, the wells 310 may be formed after receiving the receptacle 300 (Step 1220). In such embodiments, as shown in FIG. 12, the receptacle 300 may be received on the work surface with desired positions prescored or pre-marked, such aswith dimples 305 indicating desired positions for forming the wells 310. The wells 310 may be formed with the well tool 800 (FIGS. 12 and 13) by engaging the well tool 800 with the receptacle 300 (e g., pressing the well tool into the receptacle) thereby deforming the receptacle material at the indicated positions and forming the wells 310 at the indicated positions.

[0077] A population of seeds 10 is inserted into the wells 310 of the receptacle 300 (Step 1230). The population of seeds 10 may be a single seed 10 or may be a plurality of seeds 10. The seeds 10 may be inserted into a respective well 310 such that the orientation of each seed 10 is known, e g., with the crown of each seed upright or a preferred cotyledon orientation. Insertion of the seeds 10 into a respective well 310 may partially or fully affix the seeds 10 in the receptacle 300 for sampling and analysis. The seeds 10 may be inserted into the wells 310 without deforming the body 302 of the receptacle 300.

[0078] The seeds 10 may be handled or singulated from the population of seeds 10 and inserted into the wells 310 manually or by seed handling systems. Such systems are described generally herein. A detailed description of the seed singulation systems and methods contemplated herein are described in a co-pending U.S. Patent Application Serial No. TO BE INSERTED which is hereby incorporated by reference in its entirety.

[0079] In some embodiments, the seeds 10 may be inserted in the receptacles 300 through the use of automation, such as the use of a multi-axial industrial robot arm. In one example embodiment, an industrial 6-axis robot work cell is equipped with one or more of a shaker bowl, machine vision camera and light, and a custom end-effector with grips, suction cups, and / or a vacuum head to pick up individual seeds 10 from a collection of the seeds 10, and place individual seeds into the wells 310 of the receptacle 300, or push individual seeds 10 into the receptacle 300 (e.g., at pre-programmed or the dimples 305) to create the wells 310 with the seed 10 embedded therein. In some embodiments, the robot arm may include optical sensors and / or machine vision systems via which individual seeds 10 are oriented, after being picked from the collection of seeds 10, and before being placed into the receptacle 300 in the desired orientation. This may be advantageous, for example, in certain embodiments using corn seeds so that each seed 10 (or kernel) is placed in a respective well 310 with the crown exposed and the bottom of the seed 10 at the bottom of the well 310.

[0080] In embodiments, proper orientation of the seed 10 may be confirmed through use of a touch probe. The touch probe may follow the seed 10 profile to confirm orientation of the seed 10 within the well 310. For example, the touch probe may be a sensor from Gel Sight®. A user may manually check the orientation of each seed 10 with the touch probe,or the touch probe may be automated. For example, the touch probe may be coupled to the cutting device 600 or attached actuator to automatically confirm seed orientation.

[0081] In an example embodiment, a population of seeds 10 is received in a bin, and a seed 10 is singulated from the population of seeds 10 by a lifting platform coupled to the bin, the seed raised away from the remainder of the seeds 10 of the population by a suction cup at the terminal end of the lifting platform The lifting platform is sized to hold only a single seed 10, and the singulated seed 10 is rotated on the platform while an optical sensor (e.g., camera or fixed position distance sensor, etc.) captures an optical attribute of the seed 10 such as to confirm the presence of a single seed 10 on the lifting platform and / or an outside profile of the seed 10. Following analysis of the optical data received from the sensor, seed placement is carried out via a robotic arm, such as by the robotic arm picking up the seed 10 from the lifting platform and placing it in a respective well 310 of the receptacle 300.

[0082] In another example embodiment, a population of seeds 10 may be spread out on a plate or other surface, wherein a seed 10 is singulated from the population of seeds 10. In various embodiments, the seed 10 may be singulated using a variety of different methods including a lifting platform as described above, and / or other methods, including, but not limited to, a rotating disc having one or more holes or indentations configured to hold a single seed 10, e g., with vacuum. In still other examples, a seed 10 may be manually singulated from the population of seeds 10.

[0083] Continuing to refer to FIGS. 1, 2, and 15, with the population of seeds 10 inserted into a respective well 310, the seeds 10 may be positioned within the receptacle 300 (Step 1240). As the seeds 10 are positioned within a respective well 310 the seed 10 deforms the body 302 of the receptacle 300. The aligner plate 500, 550 may be used to position the seeds 10 at a known depth within their respective well 310. The aligner plate 500, 550 may be used to position the seeds 10 manually by a user or automatically by a robotic arm. Positioning the seeds 10 may concurrently partially or fully affix the seeds 10 in the receptacle 300 for sampling and analysis. In particular embodiments, where the seeds 10 are inserted into the wells 310 without a preformed tapered portion 312, positioning the seeds 10 with the aligner plate 550 may concurrently or contemporaneously form the tapered portion 312 or other internal features of the wells 310 by deforming the body 302 of the receptacle 300. In some embodiments, the seeds 10 are positioned manually (Step 1240a). Manual positioning may include a user positioning the seeds by hand. For example, the user may position the seeds 10 using their finger or a dowel to press the seeds 10 to depth. In certainembodiments, the seeds 10 are positioned by the aligner plate 500, 550 (Step 1240b). The aligner plate 500, 550 may position all the seeds 10 inserted into the receptacle 300 concurrently. The aligner plate 500, 550 may be used on its own, e.g., a user plunging the aligner plate 500, 550 by hand, or may be used with the press 570. As shown in FIG. 4, the aligner plate 500, 550 may position the seeds 10 at various positions within the well 10. For example, the seeds 10 may be positioned below the top surface 304 of the receptacle 300 in the range of 4 millimeters and 10 millimeters, e.g., 6, 7, or 8 millimeters. In embodiments, the seeds 10 may be positioned so that the crown of the seed is coplanar with the top surface 304. In other embodiments, the crown of the seed 10 may extend above the top surface 304 In still other embodiments, the seed may be pushed into a depth of the well such that a volume or clearance remains between the crown of the seed and the top surface of the well.

[0084] In some embodiments, seeds 10 are positioned in the receptacle 300 in a manner such that all the seeds 10 are substantially at the same working height. That is, each seed 10 is exposed by substantially the same amount. One advantage of aligning the seeds 10 in a common plane and to a common working height is that it facilitates use of automation equipment without the need for advanced camera vision systems or 3D profiling. In some examples, after inserting seeds 10 at varying heights (e.g., manually or using automation), the aligner plate 500, 550 may be used to reposition all the seeds 10 (e.g., pushed down) to a common working height or plane. This can be particularly advantageous in systems where the receptacle 300 is made of a deformable material. After inserting the seeds 10 in a well 310 of the receptacle 300, either manually or using automation, the aligner plate 500, 550 can be placed over receptacle 300 such that pins align with the wells 310 or markings. Pressure is then applied on the aligner plate 500, 550 causing the prongs 501, 552 to push the seeds 10 to a common depth or plane positioning the seeds 10 within the wells 310. In some embodiments, the seed 10 may be pushed into the receptacle 300 to a depth that results in a cavity being created above the crown of the seed 10 (see e.g., Step 101a in FIG. 1).

[0085] In the depicted embodiment, each well 310 provides enough displacement, compliance, grip, and compression support to reliably hold and affix the seed in place without dislodging or moving during subsequent process steps, e.g., drilling during steps 102, 202. The shape of the wells 310 or prescored dimples in the receptacle 300 are not limited to circles but could also include slots, ovals, rectangles, slits, 3 -dimensional recesses, or any other combination of geometry. In some embodiments, when using an automated system to affix the seeds 10, the wells 310 may not be pre-formed in the receptacle 300. Rather, the automated system can be programmed to affix the seeds 10 at predetermined points in thereceptacle 300, without requiring any placement indication on the surface. In such embodiments, the body 302 of the receptacle 300 may be a solid block of material, without wells 310. Alternatively, a series of dimples 305 can be used in place of wells 310, where affixing the population of seeds 10 into the receptacle 300 comprises applying a force to drive the seeds 10 into the receptacle 300 so that the seeds 10 are held in a common orientation within the dimple.

[0086] Continuing to refer to FIGS. 1, 2, and 15, in embodiments, the population of seeds 10 may be affixed for sampling and analysis after insertion or positioning within a well 310 (Step 1250). Affixing the seeds 10 may include allowing the seeds to remain within the receptable for a rest period to allow the material of the receptacle 300 to fully form around the seed 10, e.g., allowing the elastic deformation to subside. The seed 10 may rest within the well 310 with the aligner plate 500, 550 remaining coupled or mated with the receptacle 300, e.g., with the plunger 574 of the press 570 in a lowered or pressed or juxtaposed position, and with the plunger withdrawn after the rest period has transpired. In some embodiments, the aligner plate 500, 550 may be immediately withdrawn after positioning the seeds 10 within the receptacle 300. Allowing the seeds 10 to sit within the receptacle 300 undisturbed before sampling and analysis may increase the fixing force applied to the seed 10 by the receptacle 300. The rest period may allow time for any elastic deformation of the receptacle 300 induced by inserting or positioning the seeds 10 to subside, e.g., for the wells 310 to spring back towards their original dimensions. As any elastic deformation subsides, walls of the wells 310 may exert a fixing force on the seeds 10 positioned therein. The rest period may be 30 seconds, 1 minute, 3 minutes, 10 minutes, or more than 10 minutes.

[0087] In addition, the seeds 10 may be affixed by one or more of the following options: i. Using glue (such as cyanoacrylates, hot-melts, silicone, and the like). ii. Using an injection molded or 3D printed receptacle made of a pliable material, such as rubber, wherein the receptacle comprises 3 -dimensional geometry capable of fitting to a seed shape. iii. Vacuum forming a piece of thin thermoplastic around the seeds 10 in a manner similar to blister packaging, to hold the seed 10 in place. iv. Using a 2+ finger claw or grabber (e.g., similar to a screw grabber). v. Using a clamp with jaws containing a negative cavity configured to hold the seed 10 (e.g., in the shape complementary to the seed 10) being affixed to the receptacle. vi. Using a clamp made of a compliant material.vii. Using adhesive tapes. viii. Using vacuum cups shaped to conform to the seed 10, with vacuum applied. ix. Using a spring steel clip (e.g., a binder clip) or wire spring wire enclosing / wrapping the seed 10. x. Using a full plastic deformation foam material such as phenolic foam. xi. In some embodiments, instead of placing individual com kernels in the receptacle, the corn seeds may be kept on the cob and the in-seed sampling system and method may be configured to be performed directly on the cob (e.g., while rotating the cob). In such a manner, the seeds may be considered affixed (via the cob) by a mechanism configured to hold and / or manipulate the cob.

[0088] Continuing to refer to FIG. 15, the method 100, 200 of the depicted embodiment may include, at step 102, 202, creating an orifice in the seed 10 to expose a portion of non-embryonic tissue of the seed 10 (Step 1260). Generally, the orifice is created by a cutting operation such as, for example, drilling, puncturing, perforating, or otherwise cutting into the seed 10, such as through the protective seed coating. In such a manner, in various embodiments, an orifice may be considered a puncture, cut, channel, cavity, or vessel, etc. in the seed 10. As noted, such structures may be created in variety of ways, such as, for example, via puncturing, piercing, drilling, and / or any other suitable means. By creating an orifice in the seed, a solution can then be dispensed into the orifice to enable inseed sampling.

[0089] As a result of creating the orifice, in some embodiments a portion of the endosperm or cotyledon is removed. As noted, while in the depicted embodiments a cutting operation comprising a drilling operation is depicted and described, it will be appreciated that other suitable manners of creating an orifice may be used, including but not limited to, piercing, boring, cutting, grinding, abrasing, ablating or any other form of penetrating the seed coat to thereby expose the endosperm or cotyledon of the seed 10. In some embodiments, based on the configuration of the seed 10 (e.g., for a com seed) the orifice is created in the crown region of the seed 10 to expose a portion of the underlying endosperm or cotyledon area. In some embodiments, the orifice is not created in, and / or does not extend into, the embryonic region of the seed 10. By limiting the orifice to the endosperm area, viability of the seed 10 may be maintained, and the same seed 10 can be used for germination and propagation into a plant after seed sampling.

[0090] The inventors herein have recognized that by creating an orifice in the seed 10 (such as, for example, by drilling, puncturing, perforating, or otherwise cutting into theendosperm or cotyledon area to create an orifice in the seed 10), the seed 10 itself can be used for obtaining a biological sample, such as genetic sample. That is, an orifice can be created in situ in the seed 10 into which a sampling solution can be delivered for seed analysis. In this way, the need for a seed sample distinct from the seed 10 is obviated. By making the seed 10 itself the sample on which analysis can be directly performed, various issues associated with seed sample handling are overcome. For example, the need to maintain a relationship between a seed and a respective chip sample is obviated.

[0091] Optionally, the method 100, 200 may include removing a portion or all of the seed coat at the crown of each seed 10 prior to creating the orifice. In various embodiments, removing the seed coat can be done manually or automatically.

[0092] Optionally, the seed 10 may be partially or fully soaked in water or another solution that softens the seed coat, prior to creating the orifice. Presoaking the seed 10 can reduce the amount of debris generated during the creation of the orifice. In some embodiments, the presoaking can also improve the yield of biological material (e.g., DNA, RNA, protein, and / or carbohydrate) from the seed 10. In some embodiments, only the top of the seed 10 may be soaked to minimize seed coat exposure to the solution. In particular embodiments, a seed coat or pericarp removal step may be performed prior to placing the seed 10 in the receptacle 300, or while the seed 10 is in the receptacle 300.

[0093] Referring to FIG. 16, one or more of a variety of cutting devices 600 may be used to create the orifice. For example, a variety of different rotary cutting machines may be used, including, but not limited to, a drill, a Dremel® rotary tool or like device, a router, a milling machine, a cutting wheel, a coring device, etc. Such machines may employ one or more cutting tools such as, for example, drilling tools including a router bit, an endmill, or a drill bit. Other cutting tools may include a burr or the like. In certain embodiments, a cutting tool may be coupled to a CNC machine. In other embodiments, a liquid handling platform can be customized to incorporate a cutting tool. For example, a pipetting head of a liquid handling station may be replaced with a cutting tool. Still other methods may be used for creating the orifice and exposing the non-embryonic tissue including, but not limited to, laser-based cutting or ablation, use of a manual cutting device such as a blade or knife, a nichrome cutting wire, use of positive pressure from an air jet or waterjet, and / or sandblasting a portion of the seed coating and non-embryonic tissue.

[0094] The cutting tool may be selected based on seed type. Selection of the cutting tool may include, but is not limited to, consideration of seed size, seed shape, seed coat type, position of seed embryo relative to cotyledon(s), moisture content, or seed oilcontent. Further still, the cutting tool may be selected based on the size and shape of orifice desired, which itself may be a function of sample volume desired or required for the assay of a given biological material (e.g., based on whether seed DNA or seed protein is to be assayed).

[0095] As one example, the cutting tool may comprise a downcutting router bit, such as a 1 / 2-inch, 1 / 4-inch, 1 / 3-inch, 1 / 5-inch, 1 / 8-inch, or a 1 / 10-inch downcutting router bit may be used for crop seeds. As another example, an upcutting router bit, such as a 1 / 2- inch, 1 / 4-inch, 1 / 3-inch, 1 / 5-inch, 1 / 8-inch, or a 1 / 10-inch upcutting router bit may be used for crop seeds. In one particular embodiment, a downcutting or upcutting 1 / 8-inch router bit may be used for corn and soybean seeds. For example, a drill bit diameter of 0.5mm, 1mm, 2mm, 3mm, 4mm, 5mm, or larger may be drilled to a depth of 0.2mm, 0.3mm, 0.5mm, 0.75mm, 0.8mm, 0.9mm, 1mm, 1.5mm, 2mm, 3mm, 4mm, 5mm, or greater depth. As such, any diameter appropriately sized to the bounds of the seed may be used. Similarly, any drill depth may be used as long as sufficient clearance is made away from the embryo. For example, a drill bit may be used that generates a diameter that is 0.1%, 0.25%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% , or higher, and any value therebetween, of the seed diameter. As another example, a drill bit may be used that generates an orifice that extends to a depth that is 0.1%, 0.25%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or deeper (such as, for example, any depth wherein after drilling the seed maintains viability), or any value therebetween, of the seed length. In some embodiments, a downcutting bit may be preferred because its spiral design pushes the cut material downward, resulting in a cleaner shoulder while leaving a rougher bottom in the cut creating greater surface area. In other embodiments, an upcutting bit, which pulls chips from the seed up and out of the cut, may be preferred.

[0096] As noted above, the methods provided herein may be applied to a variety of different seeds, including, but not limited to, crop seeds (e g., seeds from corn (dent / field corn or sweet com), soy, sunflower, rapeseed, cotton, etc.), cereal seeds (e.g., seeds from com, barley, wheat, rice, oat, etc.), vegetable seeds (e.g., seeds from bean, brassica, cucumber, lettuce, melon, okra, pepper, spinach, squash, tomato, etc.), fruit seeds (e.g. seeds from melons including watermelon, pear, apple etc.), flower seeds, as well as tree seeds. In some embodiments, such as, for example, embodiments configured for use with some soy seeds, a 1 / 8 diameter, 140° carbide spotting drill bit may be used. In some embodiments, a shallow angle may be preferred to remove the seed coating but not much of the underlying cotyledon. In some embodiments, a 118-degree angle may be used. In some embodiments, such asembodiments configured for use with some corn seeds, an Amana 46200-K Solid Carbide Spektra™ Extreme Tool Life Coated Spiral Plunge bit may be used having 1 / 8 diameter x 1 / 2 x 1 / 4-inch shank. In some embodiments, such as embodiments configured for use with some sweet corn seeds, a downcutting bit or 140-degree bit may be used.

[0097] Continuing to refer to FIG. 16, an example embodiment of a cutting device 600 that may be used to create an orifice in each seed 10 affixed in the corresponding wells 310 the receptacle 300 is shown. In the depicted embodiment, the cutting device 600 comprises a CNC router. The cutting device 600 includes a drill bit 601 and a surface 602, each which can be customized for a receptacle 300. Any suitable rotary device like a router, a drill, or other rotary machine or the like can also be used. For example, a mini-CNC mill like the Iconic-4® produced by Axiom Tool Group, Inc may be used. The Iconic-4® has the capability of holding approximately 20 seed receptacles in the SBS microplate format (20 receptacles x 96 seeds each = 1920 seeds per deck). The Iconic-4 can be operated so as to be capable of cutting seeds up to 24,000 RPM for rapid processing, although any spindle speed may work with appropriate feed rates. CNC routers can also be utilized in parallel or in series to increase throughput or modified to include multiple gantries and spindles on a single table, or multiple spindles on a single gantry. In another embodiment, a custom multichannel drill head (for example, custom drill from vendor AutoDrill Lebanon, NJ 08833) can be manufactured and attached to a spindle to allow rapid parallel drilling (e.g., 2, 4, 6, 8, 12, 24, 48, or 96 channels). In one example embodiment, a custom deck carrier fitted to the CNC mill has matched nest positions to fit the SBS formatted seed fixturing plates. Fixture locating features such as fixed and spring-loaded locating pins (also known as spring guide pins) or spring-loaded lever arms may be used to allow the operator to secure and remove the receptacle 300 to the deck of the CNC mill without the use of tools.

[0098] In some embodiments, faster spindle speeds may improve drilling throughput (e.g., the number of seeds drilled per minute) and may provide less force imparted into the seed 10. If the spindle speed is too slow, or if the z feed rate is too fast, the seed 10 may spin out of the receptacle 300 (e.g., the foam) and / or the fixture of the seed may be lost. In such instances, the seed 10 may become damaged. In some embodiments, higher speeds may reduce tear-out of the material. For example, if around 0.75mm of material is removed from the seed 10 (only enough to expose the inner material comprising DNA, RNA, protein, and other biological molecules), high speeds may not cause the tool to slow down, which may minimize heat, and damage to the seed 10.

[0099] Returning to FIGS. 1 and 2, at step 103, 203, the method of the depicted embodiment includes removing and discarding the debris created at the orifice of each seed 10 or the population of seeds 10 by the cutting device 600. As such, the debris may include a seed fragment (e.g., seed chip, seed sample), seed dust, seed shaving, or any combination thereof based on the type of tool used for creating the orifice. For example, in certain embodiments where a drilling bit is used, the debris may include seed shavings and seed dust. In another embodiment, where a different cutting device is used for creating the orifice, the debris may include a seed fragment. In still another embodiment, where a corer is used for creating the orifice, the debris includes a seed core. Still other forms of seed debris may be formed based on the tool type, seed type, duration of sampling, location of sampling, and any combination thereof. In all cases, the debris may be defined as the seed material that is released during the creation of an orifice in the seed 10. Further, this debris includes seed material that is not used for sampling a biological trait or genotype of the seed. That is, the debris includes seed material that is thrown away before any steps related to extracting a biological sample from the seed can be performed. As a result of throwing away the debris before dispensing a sampling solution in the seed 10, contamination of in-seed sampling results with material from the debris may be averted, and sample drift or cross contamination related issues may be obviated.

[0100] Additional methods of capturing and / or removing seed dust include charged plates, ions, vacuums, pressure, dust filters, sticky traps, electrostatic precipitators, cyclone separators, baghouse filters, wet scrubbers, wet milling, wet flushing, etc.

[0101] Referring to FIGS. 15, 17 and 18, in embodiments, the method 1200 may include cleaning the drill bit 601 by plunging the drill bit 601 into the body 302 of the receptacle 300 (Step 1270). The receptacle 300 includes sacrificial regions or portions 301 for cleaning the drill bit 601. The sacrificial portions 301 may be disposed about the periphery of the body 302. In some embodiments, the sacrificial portions 301 may be between rows of wells 310. The drill bit 601 may be cleaned by plunging the drill bit 601 into the body 302 at a position along the sacrificial portion 301. Plunging the drill bit 601 into the sacrificial portion 301 may leave divots or holes in the body 302 of the receptacle 300 as shown in FIGS. 17 and 18. Plunging the drill bit 601 into the sacrificial portion 301 may knock residual dust off the drill bit 601 and reduce the risk of cross contamination. The drill bit 601 may be cleaned by plunging the drill bit 601 into the sacrificial portion 301 of the receptacle 300 between drilling of the orifice of each seed 10. In embodiments, more than one orifice is drilled between cleaning the drill bit 601. For example, 2, 4, 6, 8, 10, or morethan 10 seeds 10 may be drilled to create an orifice in the seeds 10 between plunging the drill bit 601 into the sacrificial portion 301. The drill bit 601 may be plunged into the sacrificial portion 301 with the drill bit 601 rotating in the clockwise direction, the counterclockwise direction, or not rotating. The direction of rotation or lack of rotation may be determined based on the cutting direction of the drill bit 601, e.g., upcutting or downcutting.

[0102] In one example embodiment, the debris removal is performed by a cleaning tool, e.g., a vacuum. In particular embodiments, the cleaning may be performed after the orifice has been created. In other particular embodiments, the cleaning may be performed while the orifice is being created (that is, concurrent to the drilling). In the debris removal step, the portion of the seed endosperm or cotyledon that is removed by the cutting device during the creation of the orifice is removed and discarded by the cleaning tool. While prior art seed sampling approaches require a portion of the seed endosperm to be removed, subsequently the removed portion must be handled with utmost care to ensure that sufficient biological material can be extracted from it and further that the results can be reliably correlated with the seed 10 from which the sample was taken. In the embodiments described herein, as a result of the debris removal, the orifice or well created in the seed’s seed coating, endosperm, or cotyledon region as well as the seed surface is cleaned and rendered free of any particles (e.g., endosperm or cotyledon particles) that are removed from the seed 10. This clean orifice is now the “sampling region” of the seed 10 where a solution can be delivered.

[0103] In some embodiments, by using a drilling tool to remove a portion of the seed 10 that is then discarded, various advantages may be achieved. First, a sample that is distinct from the seed 10 is eliminated. This reduces handling issues resulting from the small size of the sample such as sample drift. In particular, since the debris is discarded, the possibility of sample drift from one well to another is averted. As another example, the reliance on complex sample management and tracking systems (e.g., LIMS), for ensuring that the identity of the portion removed from the seed 10 is correctly correlated with the identity of the corresponding seed 10 during all subsequent processing steps, is substantially reduced.

[0104] Additionally referring to FIG. 19, in some embodiments, a vacuum source is used to remove debris from each orifice, as well as any debris from the receptacle 300 (e.g., into a dedicated container). In embodiments, the debris may be removed using canned or forced air, or a positive air flow, which direct the debris away from the seeds 10 and the receptacle 300. The forced air may be used to direct the debris towards a vacuum source where the debris may be collected into a dedicated debris container. In some embodiments,the vacuum may be positioned adjacent to (e.g., coaxial or coplanar to) the drilling tool such that the debris is removed while the drilling tool is creating the orifice in the seed 10. One example of such an embodiment is shown at FIG. 19, a CNC router 700 is customized to incorporate both a drill head 701 and an associated cleaning tool 703, depicted herein as a vacuum head. In one example, the vacuum head is the Iconic-4 dust-shoe that is modified to be fitted to a standard CNC router mounting attachment. Such a combined drilling and vacuum tool allows for simultaneous drilling and debris removal, ensuring that seed debris from the drilling of a given seed 10 does not interfere with later analysis of seed material. In other words, a clean orifice can be provided wherein the seed sampling region of a given seed 10 is free of any self-contamination from seed debris belonging to the same seed 10 as well as cross-contamination from seed debris belonging to any other seed 10. In other embodiments the drilling tool aids in the cleaning of the orifice by creating a positive pressure locally during operation of the tool, thereby blowing the debris away from the seed 10. This also enhances the integrity of the subsequent molecular analysis. In still other embodiments, the combined drilling tool and cleaning tool may be included as part of an automated robotic arm or gantry system. In other embodiments, a dust shoe may be used, which is configured to trap and remove seed debris. In some embodiments, the dust shoe may include a plurality of bristles and / or the dust shoe may be connected to a vacuum source. In other embodiments, the dust shoe may not be connected to a vacuum source. Some embodiments may further include a secondary cleaning step to clean and remove seed dust from the dust shoe. In certain embodiments, the dust shoe may have forced air nozzles blowing out wells 310 that are positioned adjacent to the well 310 being milled, with a vacuum source to capture debris causes from milling and forced air debris.

[0105] Alternative cleaning methods may include, but are not limited to, heatbased cleaning (e.g., induction, nichrome wire, or flame), use of a liquid stream, use of mechanical abrasion (e.g., a brush, sponge, or eraser), use of an ultrasonic liquid bath, soaking of at least the drilling tool and / or the drilled seed 10 in water or a chemical bath (e.g., detergent / surfactant), use of ultraviolet generated ozone, or use of a sacrificial consumable (e.g., disposable foam or paper abrasive product to mechanically knock off debris while not substantially dulling the bit). Additional cleaning methods may include Chemical cleaning, Bubble cleaning, Ultrasonic cleaning, Heat / temperature cleaning, Detergent cleaning, Plasma cleaning, Cold plasma cleaning, Radiation cleaning, Irradiation cleaning, pH-based cleaning (acids / bases), Vacuum cleaning, Pressure cleaning, Electrochemical Advanced Oxidation Process (EAOP) cleaning, Laser cleaning, Water cleaning, Air cleaning, Consumableabrasion cleaning, UV (Ultraviolet) cleaning, Solvent cleaning, DNAse, protease, or chelation agents. An appropriate cleaning tool, as known in the art, may be used based on the selected cleaning method.

[0106] In some embodiments, additionally or optionally, a drilling tool may be plated with a low friction or non-stick coating (e.g., Titanium Nitride or Nickel PTFE) to reduce sticking of the debris to the drilling tool and associated contamination or carryover. Select CNC mills may be equipped with automatic tool changers that allow the machine to drop-off and pick-up new cutting tools after they were soiled or have had too much wear. Further still, the CNC mill may be configured with multiple drilling tools in a configuration that matches the number and placement of the wells 310 in the receptacle 300 so that each seed is drilled by a distinct drill. A common vacuum tool, or dedicated vacuum tools coupled to each drilling tool, may be operated to clean the debris. In some embodiments, a sleeve may be provided around the drilling tool (e.g., around the drill head 701 of FIG. 19), wherein the sleeve is made of a material with an affinity to hold debris via a static charge, with or without the presence of an electric or magnetic field, or via Van der Waals forces. The sleeve may be used to capture the removed material. Additional drill coatings may include, but are not limited to, Titanium Nitride (TiN), Titanium Carbonitride (TiCN), Aluminum Titanium Nitride (AlTiN), Titanium Aluminum Nitride (TiAlN), Diamond-like Carbon (DLC), Black Oxide, Cobalt, Zirconium Nitride (ZrN), and Teflon®.

[0107] Referring back to FIGS. 1 and 2, next, at steps 104, 204, the method includes applying a solution to the exposed portion of the cotyledon or endosperm, thereby forming an in-seed soak solution directly in the orifice in the seed 10. By pipetting the solution into the orifice multiple times, the soak solution comprises one or more extracted biological molecules such as, e.g., protein, DNA, RNA, carbohydrate, and / or glycan from the seed 10. Since the solution is applied directly into the orifice to contact the exposed portion of the seed 10, the need for a dedicated soaking tray or corresponding soaking space in the receptacle 300 is averted. In addition, since the drilled seed 10 itself is now the source of the biological material, instead of a removed portion of the seed 10, the need for complex LIMS systems for tracking the results of an analysis of the solution are reduced. In some embodiments, as described above, the tapered portion 312 of the wells 310 may function as an ex-seed soaking region. The tapered portion 312 of the well 310 may allow for a greater volume of seed soak solution to be dispended for each seed 10. This may yield a greater volume of analyzable biological information from each seed 10.

[0108] In one embodiment, the solution dispensed into the orifice is an alkaline solution used for extracting nucleic acids from the seed endosperm, such as DNA and RNA. The alkaline solution may comprise any known alkali suitable for DNA extraction, such as NaOH, KOH, etc. In other embodiments, the solution dispensed into the orifice may be a solution used for extracting nucleic acids, proteins, carbohydrates, lipids, or other biological material from the exposed part of the seed 10, e.g., the endosperm. In some embodiments, based on the compatibility of the solutions, one or more different extraction solutions may be sequentially dispensed to allow for sequential extraction of material (e.g., a first solution used for extracting DNA followed by a second solution used for extracting protein, etc.) Still other solutions that can be dispensed include an enzyme solution, water, or any other suitable solution that will extract an indicator of a trait or characteristic of interest.

[0109] An example embodiment of a solution used for extracting DNA from the exposed non-embryonic seed tissue is an alkali solution, such as the alkaline solution of U.S. Patent No. 10,011,828 (the ’828 patent). The alkali solution may, in some instances, be sodium hydroxide (NaOH), or potassium hydroxide (KOH), or other alkali solutions. Unlike the approach of the ’828 patent, which requires the entire seed 10 to be soaked in the extraction solution, following a pre-treatment step, the presently disclosed embodiments are able to perform the extraction by exposing only a portion of the seed 10 to the solution in one step. In addition to reducing the volume of solution required to achieve DNA extraction (since the whole seed 10 is not soaked), without compromising DNA yield or quality, the approach of the presently disclosed embodiments reduce exposure of the seed embryo to the chemicals of the alkaline solution, improving germination efficiency and viability. Further, DNA extraction can be performed in a simple manner, without the need for pre-treatment of the seed 10. Furthermore, DNA can be extracted from the seeds 10 where the entire seed coat would otherwise be prone to damage or sloughing due to exposure to the alkaline solution.

[0110] Other DNA extraction solutions may be dispensed including but not limited to solutions comprising detergents (e.g., SDS, Tween20, EDTA, CTAB, PVP, chelating agents), enzymes, alcohol additives (e.g., PEG), etc. Further still, the extraction solution and protocol may be modified to include known methods of DNA extraction such as through the use of magnetic beads, alcohol precipitation, etc. In certain embodiments, the alkaline solution includes NaOH solutions comprising at least 5mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM, 150 mM, or 200mM NaOH.

[0111] In some embodiments, optionally, a volume of water or alkali lysate soaking solution can be added to the orifice and allowed to incubate for a duration to increase the yield of biological material extracted (e.g., to increase the DNA or RNA yield). Herein, the volume can be a minimal volume that is sufficient to cover the bottom of the orifice, or it can be the entire volume of the orifice. In one example, 15 pl of water or alkali lysate is incubated in the orifice for 5 minutes or longer to increase DNA yield.

[0112] In some embodiments, such as where the receptacle 300 is made of a deformable material, after an orifice has been drilled into the affixed seed 10 and the debris removed, and prior to dispensing the solution into the orifice, the seed 10 may be pushed deeper into the well 310 of the receptacle 300, e.g., into the stem portion 318, so additional seed soak solution can be dispensed and accumulated. This may be advantageous in applications where biological material yield can be limited.

[0113] The inventors have found that the method disclosed herein produces a surprising level of DNA yield. Moreover, some examples (such as those shown in the Example section) show an increase in DNA yield obtained through the disclosed method.

[0114] Continuing to refer to FIGS. 1 and 2, at step 105, 205, the method 100, 200 includes removing the seed soak solution and transferring the solution to a container having a fixed relationship with the seed 10 or the position of the seed 10 in the receptacle 300. For example, seed drilling, debris removal and in-seed soaking may be performed for each seed 10 in a first receptacle 300 and then the seed soak solution from each extraction may be transferred to the corresponding wells of a second receptacle 300 where each well 310 of the second receptacle 300 has a fixed relationship with the wells 310 of the first receptacle 300 . In this way, the results of an analysis of the soak solution can be tracked and correlated with the corresponding source seed 10.

[0115] In embodiments, each receptacle 300 and soak solution container has an identifier 303, such as a barcode or human readable label, saved in a database for tracking purposes. In particular embodiments, each well 310 has an identifier 303 for tracking purposes. After transfer of the soak solution from the receptacle 300 to the soak solution container, the drilled but viable seeds 10 may be stored in the receptacle 300 until the results (Steps 106A, 106B, 206) of an analysis of the soak solution is completed.

[0116] In one example embodiment, an alkali solution comprising 20mM NaOH is applied to the orifice in the seed 10. Further, the solution may be dispensed, and the soak solution extracted multiple times to provide a larger volume (e.g., for multiple types of assays) or for a greater DNA yield. For example, approximately 15 pl to 20 pl of solutionmay be initially pipetted into the orifice and 15 pl to 20 pl of solution may be transferred to an assay plate. Then, another 15 pl to 20 pl of solution may be pipetted into the orifice and 15 pl to 20 pl of solution may be again transferred to the assay plate (thereby providing 30 pl to 40 pl of soak solution from the same seed). This same process may be iterated multiple times. Additionally or alternatively, an additional well space can be created in the receptacle 300 above the exposed seed 10 by pushing the seed 10 down in to the well 310 so that a larger volume of seed soak solution can be generated. For example, the seed 10 may be entirely disposed within the stem portion 318 of the well 310.

[0117] In certain embodiments where the alkali solution used for extracting DNA from the seed 10 is too basic (alkaline) to be compatible with downstream processes such as downstream PCR Taq polymerase reactions or sequencing reactions, a buffer or acid (e.g., TRIS HCL or acetic acid) may be added to neutralize the solution to an acceptable pH. In certain embodiments, the seed soak solution can be removed from the seed 10 by pipetting or other suitable means including but not limited to pumping or capillary actions. The transfer may be done by manual pipette, or by a multi-channel pipetting automation equipment (such as the Tecan Freedom Evo MultiChannel Arm™ MCA 96).

[0118] To improve pipetting performance, a hydrophobic coating / spray may be pre-applied to the seed 10 before drilling the orifice (Step 102, 202). Addition of a hydrophobic coating can help contain the droplet (and larger droplets) of the solution or soak solution during the pipetting and mixing step and ensure that the droplet of soak solution does not fall outside of the seed orifice or get contaminated with maternal DNA from contact with the seed coat or be irrecoverably lost on the receptacle 300.

[0119] With continued reference to FIGS. 1 and 2, at step 106, 206, the method includes analyzing the extracted seed soak solution for the presence or absence of a characteristic or trait of interest. Analysis may be done using a Polymerase Chain Reaction (PCR) analysis. The DNA can be dispensed directly into a 96, 192, 384, or 1536 well PCR plate with PCR chemistry, or tape with PCR chemistry and can be run directly on a plate based PCR system (such as the QuantStudio™ 6 Flex Real-Time PCR System). Other DNA or RNA analysis methods could also be used, such as isothermal amplification or variations of next generation sequencing (NGS).

[0120] The disclosed methods, apparatus and systems are also configured to be compatible with existing and upcoming DNA sequencing-based technologies, such as Skim sequencing and Genotyping by Sequencing (GBS), including tunable Genotyping by Sequencing (tGBS), due to the need for small DNA liquid volumes (about 4-10pl, dependingon pre-QC steps, for multiple assays). The methods and apparatus and systems are also compatible with legacy genotyping assays like Taqman or KASP, where higher volumes of DNA and / or DNA concentration can be required.

[0121] In other embodiments, the protein, carbohydrate, or oil profile of the seed 10 may be analyzed by assaying the soak solution and inferring the presence or absence of characteristic traits in the seed 10. As non-limiting examples, DNA analysis may be performed to identify the presence of a transgene, vector, extrachromosomal component, an allele, a haplotype, a gene edit, an inversion, a deletion, an insertion, or a mutation in the genome of the seed 10.

[0122] Continuing to refer to FIGS. 1 and 2, in step 107, 207, the method includes identifying or selecting seeds 10 of interest based on the analysis. The selected seeds 10 are then moved forward for germination and propagation into viable plants that are introduced into breeding pipelines or research plots. The seeds 10 of interest can be ejected from the receptacle 300, e.g., manually by an operator or by using automation. In some embodiments where the receptacle 300 is made of a deformable material, following analysis, an ejecting device may be configured to push the seeds 10 through the receptacle 300 (such as a device comprising a flat ended tool in the collet of the cutting device 600) and configured to eject the seeds 10 directly onto a sowing container (such as a clamshell or seed packet) or growth medium, or a growing container comprising growth medium. In some embodiments, the CNC device used for drilling the seeds 10 may be modified so that the same equipment can be used for drilling and ejecting or selecting the seeds 10 of interest. In certain embodiments, the seeds 10 may be selected or ejected using an industrial robot arm with a custom end effector. In embodiments, where a first CNC device is used for creating the orifice, a second CNC device may be provided in series with the first CNC device, to serve as the ejecting device.

[0123] In another embodiment, the receptacle 300 may have a hole feature in the bottom of the receptacle 300 which allows the seeds 10 to be ejected from the receptacle 300 by an ejector pin. A hole in the bottom of the receptacle 300 may not be needed if the receptacle 300 is composed of foam material that allows the seed 10 to be pushed through the body 302 of the receptacle 300 without damaging the seed 10 or disrupting neighboring seeds 10. If an industrial robot arm is used, the same robotic work cell may be used for both seed 10 loading and seed ejection by use of a dual end effector on the robot to allow for both processes.

[0124] In some embodiments, to reduce consumables, once liquid handling on the receptacle 300 is completed and the seeds 10 have been selected, the receptacle 300 can be cleaned and sterilized for reuse. Once the soak solution analysis is complete, the seeds 10 of interest may be sorted away from the others, while the unwanted seeds 10 are culled.

[0125] Referring now to FIG. 20, another embodiment of an in-seed sampling method 900 is disclosed, with reference to the receptacle 300 and the aligner plate 500, 550 of FIGS. 3-11, implementing what may be referred to as the “bird bath method.” In the method of FIG. 20, at step 901 the seeds 10 are first affixed into respective wells 310 of a first receptacle 300 in a first orientation. At step 902, inner seed material is exposed by a cutting machine or alternatively by manually cutting the crowns of the seeds 10, which may preferably be water soaked for manual methods. At step 903, debris is removed from the orifice. Then, at step 904, following creation of an exposed orifice, the seeds 10 are placed in a receptacle 300 in a second, different orientation to enable sample solution application and seed soak solution extraction. In certain embodiments of the method 900, the seed 10 can be initially inserted into a first receptacle 300 in a first orientation with the crown of the seed 10 exposed, as previously described, and then, following drilling and debris removal, the seed 10 may be removed from the receptacle 300 and re-inserted into the same receptacle 300 or a second receptacle 300 in a second orientation, different from the first orientation, for solution application. The first or second receptacle 300 may have wells 310 with only a tapered portion 312 and no stem portion 318. The second orientation may be opposite to the first orientation with the crown facing the bottom of the well 310 and the tail or bottom portion of the seed 10 extending out of, or co-planar to, the top surface 304 of the receptacle 300. A robotic arm comprising an associated vision system may be configured to initially place the seed 10 in the receptacle 300 in the first orientation and then subsequently remove and flip each seed 10 to the second orientation. At step 905, the first solution is applied to the well 310 and in contact with the created orifice to form an in-seed soak solution comprising biological material extracted from the exposed portion of the seed 10. At step 906, the seed soak solution is removed from the well 310 and analyzed, and at step 907, the seeds 10 of interest are selected based on the results of the analysis for further germination and propagation.

[0126] In various embodiments, DNA yield may be a function of surface area. Full seed soaking for corn seeds may provide more DNA yield since it allows for more area to be exposed, up to the far edges of the crown. For the full seed soak method com seeds may be pre-soaked in water to allow easy cutting (and greater DNA yield). When seeds arehand cut and pre-soaked more DNA may be obtained, but a tradeoff may be dealing with releasing DNA from the maternal seed coat that may not be representative of the endosperm and can make the analysis difficult or impossible. It is possible to soak only the top cut of the corn seed to minimize seed coat exposure - such as the bird bath method described in method 900.

[0127] With regard to soy seeds, it may not be possible to fully soak soy seeds without losing the seed coating which provides structural support to the seed. Without a seed coating the dicot seed can crack or split unless handled delicately. If extensive splits or cracks have occurred, the embryo and cotyledons may be severely disrupted or destroyed, and it may be unlikely that the seed will be able to germinate successfully. The damaged seed may lack the necessary internal structures and resources to support the growth of the seedling. When the soy seed coat falls off the seed when soaked, it rapidly expands, effectively causing the seed coat to become a loose bag around the seed, resulting in the seed not having structural support for sowing.EXAMPLES

[0128] The following Examples are illustrative only and do not limit the scope of the present disclosure or the appended claims.Example 1 : Seed Analysis Protocol

[0129] Materials: Custom phenol or polyisocyanurate foam block with 96 target positions or holes for 9mm pitch according to SBS plate standard, approximately 1” deep. 20mM of NaOH solution. 0.5M TRIS solution. lOOpl PCR plate (e.g., #4ti-0960 FrameStar® 96 Well Skirted PCR Plate).

[0130] Equipment and tools: 96 channel liquid handler, mini mill with vacuum collection and block fixturing, mill tooling / drill bits (Soy: 1 / 8 0 140° Carbide Spotting Drill; Com: Amana 46200-K Solid Carbide Spektra™ Extreme Tool Life Coated Spiral Plunge 1 / 8 Dia x 1 / 2 x 1 / 4 Inch Shank ), plate centrifuge (e.g., Thermo Scientific™ Sorvall™ Legend™ XF Centrifuge), custom press tool (to push all seeds down to same vertical / Z depth).

[0131] Procedure: Load seeds on 9mm pitch into 96 positions (by hand, with vacuum manifold with suction cups, or by other automated means). Push all seeds down to same vertical position so the tops of the seeds, regardless of seed length, are all on the same plane. Mill large surface of seed to create an orifice while vacuuming out all debris (approximately 0.75mm deep). Any visible leftover debris may inhibit PCR due to inhibitors (proteins, phenols, etc.), cause well-to-well differences, and associated data quality issues.Using a liquid handler, such as a Tecan Evo 150 MCA equipped with a 96-channel head, dispense 70pl of 20mM NaOH and mix the solution 20 times to form a homogenous seed soak solution. Transfer the seed soak solution to a new 96 well PCR plate. Spin the plate in centrifuge to 4000 RPM and transfer 32ul or greater to a new 96 well PCR plate. If greater yield is desired, incubate at 65°C overnight or 3 hours at 95°C. Neutralize the pH by adding 1 pl of 0.5M TRIS Hydrochloride per lOpl of soak solution. The DNA soak solution is now ready for PCR and / or tGBS library prep. Typically less than 20pl of material is needed to run many thousands of markers on tGBS. Genotyping platforms such as Nexar require approximately 1 pl per assay and would require more DNA yield. Both corn and Soy samples were run using the above method utilizing Agriplex tGBS analysis with success.Example 2: PCR test determining DNA yield on corn seed

[0132] FIG. 21 is a real-time amplification plot of an example test to determine DNA yield. A real-time PCR test run was performed on a com seed with 175mM NaOH, dispensing 17pl on the seed orifice, 4x times with 20 mixes, then centrifuged, and transferred to a new plate with 68 pL of lOOmM TRIS Hydrochloride. A downcutting bit with a 1 / 8” bit was used to expose the endosperm of the corn seed. The real-time PCR test determines the relative DNA yield and quality. Multiple variables were tested to determine the optimal molarity of the alkaline solution and neutralizing buffer. The plot is a real-time or qPCR plot showing fluorescent signal on the Y axis and PCR cycle on the X axis. During the PCR reaction a heated and cooled TAQ enzyme replicates the DNA region of interest and enables a fluorescent signal.

[0133] Signals read after each cycle and the signal is recorded, allowing a user to indirectly quantify how much DNA exists in the sample. This approach was used to determine how much DNA was harvested.

[0134] A second variable that was successfully used was 20mM NaOH, comprising 15 pl soak, 20 mixes, and 15 pl water. Because the molarity of the NaOH lysate buffer is so low, no TRIS Hydrochloride buffer is required to neutralize the pH for the PCR reaction (and associated Taq polymerase enzyme).

[0135] FIG. 22 is a PCR plot of an example test utilizing the soak solution to determine the presence of alleles an endpoint PCR test. An endpoint PCR reaction test was run on the same com seed as shown in FIG. 21, with 175mM NaOH of DNA with 20 mixes and lOOmM TRIS, using a downcutting 1 / 8” bit. The PCR endpoint reaction has a multiplex assay which either amplifies an allele from the father, mother or one of both (heterogenous[HET] is shown in the middle cluster). A large number of samples were placed in a water bath and thermocycled in bulk, before being put on a reader to read the fluorescent signal for allele 1 (i.e., grouping 1310 in plot 1301) or allele 2 (i.e., grouping 1320 in plot 1301).FIG. 23 is a PCR plot of an example test determining DNA clustering performance for homogeneous (1310, 1320) and heterogeneous (1330) allele clusters using a different SNP assay. An endpoint PCR reaction test was run on the same com seed as shown in FIG. 21, with 17pL 175mM NaOH of DNA with 20 mixes and 17pL lOOmM TRIS Hydrochloride, upon a seed exposed using a downcutting 1 / 8” bit. The PCR endpoint reaction has a multiplex assay which either amplifies the presence an allele on either the X or Y axis, or combination of both. A number of samples were placed in a water bath and thermocycled in bulk, before being put on a reader device to read the fluorescent signal for allele 1 (grouping 1310 in plot 1302), allele 2 (grouping 1320 in plot 1302), or a mix of alleles forming a heterogenous cluster (grouping 1330 in plot 1302).Example 3 : PCR test determining DNA yield on soy seed

[0136] FIG. 24 is a PCR plot of an example test determining DNA cluster quality as shown in graph 1600. An endpoint PCR reaction test was run on a soy seed population, with 20pl DNA with 100 mM NaOH seed soak solution with 20 mixes and 40pl TRIS Hydrochloride to neutralize the pH, using a upcutting 140-degree point angle and 1 / 8” bit. The PCR endpoint reaction shows a multiplex assay amplifying the presence of one or both alleles. A number of samples were placed in a water bath and thermocycled in bulk on an F4 population showing segregating materials with a heterogeneous cluster (center). The samples were put on a reader to read the fluorescent signal for allele 1 (grouping 1610 in plot 1600), allele 2 (grouping 1640 in graph 1600), a heterogenous cluster of both alleles (grouping 1630 of plot 1600) and indeterminate outliers (grouping 1620 in plot 1600).

[0137] Many modifications and other implementations of the disclosure will come to mind to one skilled in the art to which this disclosure pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosure is not to be limited to the specific embodiments disclosed herein and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.Example 4: Seed Analysis Protocol

[0138] Custom phenol or polyisocyanurate foam block with 96 target positions or holes for 9mm pitch according to SBS plate standard, approximately 1” deep. 70pL of 20mM NaOH solution. For trait assay 175 pL of lOOmM NaOH solution. 0.5M TRIS solution. 100 mM TRIS HCL. lOOpl PCR plate (e.g., #4ti-0960 FrameStar® 96 Well Skirted PCR Plate).

[0139] Equipment and tools: 96 channel liquid handler (Tecan Evo 150 MCA), mini mill with vacuum collection and block fixturing (Axiom Iconic CNC mill), mill tooling / drill bits (Soy: 1 / 8 0 140° Carbide Spotting Drill; Corn: Amana 46200-K Solid Carbide Spektra™ Extreme Tool Life Coated Spiral Plunge 1 / 8 Dia x 1 / 2 x 1 / 4 Inch Shank ), plate centrifuge (e g., Thermo Scientific™ Sorvall™ Legend™ XF Centrifuge), custom press tool (to push all seeds down to same vertical / Z depth).

[0140] Procedure: Load seeds on 9mm pitch into 96 positions using a vacuum manifold tool (single channel or multi-channel) to pick the seeds and place them into the foam block. Push all the seeds down to the same height, e.g., approximately an 8mm depth, using a pin tool or a custom press. Mill large surface of seed to create an orifice (approximately 0.75mm deep) while vacuuming out all debris using an Axiom Iconic CNC mill with a spring fixture to hold the foam blocks and a spring-loaded vacuum device to pin down the foam block flat while milling and removing debris. Dispense NaOH solution of 70pL of 20mM or 175pL of lOOmM for trait assays. The solution may be dispensed using a liquid handler, such as a Tecan Evo 150 MCA equipped with a 96-channel head. Allow NaOH solution to sit on seeds for an incubation time, e.g., approximately 5 minutes, at room temperature, e.g., 20°C to 25°C. Where 70pL 20mM NaOH solution is dispensed the incubation time may foregone. Where 175pL of lOOmM NaOH solution is dispensed the incubation time of 5 minutes may yield more DNA or other biological information. Wash the seeds by adding water, e.g., approximately 150pL of water, discarding the water wash solution, and then dry the seeds in moving air while still in the foam block, e.g., for 12 hours or longer. The seeds may be washed two or more times to fully remove the NaOH solution from the seeds to minimize risk of stunted seed germination. Polymers such as polyethylene glycol (PEG) or polyacrylamide (PAM) may be added to the seeds after soaking to improve germination. Transfer the seed soak solution to a new 96 well PCR plate. Mix the solution in the PCR plate 20 times to form a homogeneous seed soak solution. Mixing may be performed by the liquid handler. Spin the PCR plate in a centrifuge to 4000 RPM using a Thermo Scientific™ Sorvall™ Legend™ XF Centrifuge for approximately 1 minute. Transfer 32pL-100pL, or more, of the seed soak solution to a new 96 well PCR plate using the liquid handler. Optionally, if greater yield is desired, incubate the seed soak solution at65°C overnight or for 3 hours at 95°C. Neutralize the NaOH solution using TRIS HCL. Where 70pL at 20mM of NaOH solution is used, neutralize with I L at 0.5 mM TRIS HCL per lOpL of NaOH seed soak solution. Where lOOpL at 20mM of NaOH solution is used, neutralize with 55 pL of TRIS HCL solution at lOOmM per 40pL of supernatant (a 11 :8 ratio of TRIS HCL to supernatant). The DNA soak solution is now ready for PCR and / or tGBS library prep. Typically less than 20pl of material is needed to run many thousands of markers on tGBS. Genotyping platforms such as Nexar require approximately 1 pl per assay and would require more DNA yield.Example 5: Seed analysis protocol for certain seed types such as soy seeds

[0141] Custom phenol or polyisocyanurate foam block with 96 target positions or holes for 9mm pitch according to SBS plate standard, approximately 1” deep. 70pL of 20mM NaOH solution. For trait assay 175 pL of lOOmM NaOH solution. 0.5M TRIS solution. 100 mM TRIS HCL. lOOpl PCR plate (e.g., #4ti-0960 FrameStar® 96 Well Skirted PCR Plate).

[0142] Equipment and tools: 96 channel liquid handler (Tecan Evo 150 MCA), mini mill with vacuum collection and block fixturing (Axiom Iconic CNC mill), mill tooling / drill bits (Soy: 1 / 8 0 140° Carbide Spotting Drill; Corn: Amana 46200-K Solid Carbide Spektra™ Extreme Tool Life Coated Spiral Plunge 1 / 8 Dia x 1 / 2 x 1 / 4 Inch Shank ), plate centrifuge (e.g., Thermo Scientific™ Sorvall™ Legend™ XF Centrifuge), custom press tool (to push all seeds down to same vertical / Z depth).

[0143] Procedure: Load seeds on 9mm pitch into 96 positions using a vacuum manifold tool (single channel or multi-channel, e.g., 8 channel) to pick the seeds and place them into the foam block. Push all the seeds down to the same height, e.g., approximately an 8mm depth, using a pin tool or a custom press. Allow the press to hold the seeds at depth for approximately 1 minute to ensure the seeds are positioned at an even height. Install Foam block onto mill and mill away surface of each seed to create an orifice (approximately 0.5mm deep) and while vacuuming out all debris created during milling. Dispense 175 pl of lOOmM NaOH lysate to top seeds using 96 channel liquid handler and incubate at room temperature for 10 minutes. Transfer lOOpl of lysate in the foam block to a new 96 well PCR plate. Spin the PCR plate in a centrifuge to 4000 RPM for approximately 5 minutes to urge loose debris to the bottom of the PCR plate. After spinning, transfer 40 pl of supernatant to a new PCR plate and add 55 pl lOOmM TRIS HCL pH 7.4 to neutralize the NaOH solution. Refrigerate the neutralized seed soak solution for storage until genotype testing is performed. Test the genotype of the sampled seeds. Dry the seeds in the foam blocks overnight using moving air,e.g., with a box fan, or with an incubator. Select seed of interest based on the genotyping results. Dispose of unused seeds and foam block according to proper disposal procedures.

Claims

WHAT IS CLAIMED:

1. A receptacle for affixing a population of seeds, the receptacle comprising: a body having a top surface and bottom surface opposite the top surface, the body defining a well in fluid communication with the top surface, the well having a mouth disposed on the top surface and a tapered portion comprising one or more walls extending away from the mouth into the body and towards the bottom surface, the well configured to receive and affix a seed of the population of seeds therein.

2. The receptacle according to claim 1, wherein the one or more walls of the well are configured to exert a fixing force on the seed to hold the seed at a predetermined position and a known orientation within the well.

3. The receptacle according to claim 2, wherein the body is configured to deform when the well receives the seed therein.

4. The receptacle according to claim 3, wherein the body is configured to elastically deform in response to the seed being received within the well, the tapered portion configured to increase the fixing force exerted on the seed as elastic deformation of the body subsides.

5. The receptacle according to claim 1, wherein the tapered portion slopes inwardly away from the mouth at an angle between approximately 5 degrees and approximately 45 degrees from vertical.

6. The receptacle according to claim 5, wherein the one or more walls comprises one wall and the tapered portion of the well has a frustoconical profile.

7. The receptacle according to claim 1, wherein the well includes a stem portion in fluid communication with the tapered portion, the stem portion extending from an apex of the well towards the bottom surface of the body, the apex spaced apart from the mouth by the tapered portion.

8. The receptacle according to claim 7, wherein the stem portion has a cylindrical profile.

9. The receptacle according to claim 7, wherein the tapered portion has a first diameter and a second diameter, the first diameter equal to a diameter of the mouth and the second diameter equal to a diameter of the stem portion.

10. The receptacle according to claim 7, wherein the stem portion is in fluid communication with the bottom surface such that the well extends entirely through the body and the top surface and the bottom surface are in fluid communication therethrough.

11. The receptacle according to claim 1, wherein the tapered portion has a circular crosssection.

12. The receptacle according to claim 1, wherein the tapered portion has an oblong crosssection.

13. The receptacle according to claim 1, wherein the mouth is spaced apart from the top surface such that the well is defined to include a head space between the top surface and the mouth.

14. The receptacle according to claim 1, comprising a plurality of other wells forming an array of wells with the well, each well of the array of wells configured to receive and affix a respective seed of the population of seeds.

15. The receptacle according to claim 14, wherein the wells of the array of wells are distributed uniformly across the top surface of the body.

16. The receptacle according to claim 1, wherein the body is made of a deformable material.

17. The receptacle according to claim 16, wherein the deformable material is a polyisocyanurate foam or a phenolic foam.

18. The receptacle according to claim 16, wherein the foam material has a density of approximately 2 lbs / ft3.

19. The receptacle according to claim 1, wherein the body is formed as a monolithic construction.

20. A system for affixing a population of seeds comprising: a receptacle according to claim 1; and an aligner plate configured to position the seed within the well at a known depth.

21. An apparatus for affixing a population of seeds, the apparatus comprising: a receptacle having a top surface and bottom surface opposite the top surface, the receptacle defining a well in fluid communication with the top surface, the well having a mouth disposed on the top surface and a tapered portion comprising one or more walls extending away from the mouth into the receptacle and towards the bottom surface, the well configured to receive a seed of the population of seeds to hold and affix the seed therein; and an aligner plate configured to position the seed within the well, the aligner plate including a prong that positions the seed within the well at a known depth, the seed elastically deforming the receptacle when the seed is positioned such that the well exerts a fixing force on the seed as elastic deformation of the receptacle subsides.

22. The apparatus according to claim 21, wherein the tapered portion of well slopes inwardly from the top surface at an angle between approximately 5 degrees and approximately 80 degrees from vertical.

23. The apparatus according to claim 22, wherein the tapered portion is configured to increase the fixing force exerted by walls of the well on the seed to hold the seed at a predetermined position and a known orientation within the well.

24. The apparatus according to claim 23, wherein the tapered portion is configured such that the fixing force increases as elastic deformation of the receptacle subsides.

25. The apparatus according to claim 21, wherein the receptacle comprises a plurality of other wells forming an array of wells with the well, each well of the array of wells configured to receive a respective seed of the population of seeds to hold and affix each seed.

26. The apparatus according to claim 25, wherein the aligner plate is configured to position each seed of the population of seeds at a known depth simultaneously.

27. The apparatus according to claim 26, wherein the aligner plate comprises a plurality of other prongs forming an array of prongs with the prong, each prong of the array of prongs configured to position a respective seed within a respective well such that each seed elastically deforms the receptacle when the aligner plate positions each seed within a respective well.

28. The apparatus according to claim 26, wherein the aligner plate is configured to position each seed of the population of seeds at the same depth within a respective well relative to the other seeds of the population of seeds.

29. The apparatus according to claim 26, wherein the aligner plate is configured to position each seed of the population of seeds at the same depth within in a respective well such that the crown of each seed coplanar with the crown of the other seeds of the population of seeds.

30. A method of affixing a population of seeds, the method comprising: inserting a seed of the population of seeds into a well of a receptacle, the well having a tapered portion comprising one or more walls; and positioning the seed at a known depth within the well such that the tapered portion exerts a fixing force on the seed.

31. The method according to claim 30, comprising allowing the seed to remain in the receptacle undisturbed for a rest period prior to subjecting the seed to an analysis process.

32. The method according to claim 30, wherein positioning the seed includes using an aligner plate to dispose the seed at the known depth.

33. The method according to claim 32, wherein the aligner plate elastically deforms the tapered portion contemporaneously when positioning the seed.

34. The method according to claim 30, wherein positioning the seed includes disposing the seed at a depth equal to a depth of another seed of the population of seeds inserted into another well of the receptacle such that the crown of each seed is coplanar with one another.

35. The method according to claim 30, wherein inserting the seed includes orienting the seed with respect to the well.

36. The method according to claim 35, wherein inserting the seed in the well is performed manually.

37. The method according to claim 35, wherein inserting the seed is automated.

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