Automated system for removing tissue samples from seeds and related methods

Abstract

A seed sampling system is provided that includes an automated seed load assembly operable to singulate seeds from a plurality of seeds or to load individually stored seeds and an automated seed sampling assembly including at least one sampling module operable to remove a tissue sample from one of the singulated seeds. The system further includes an automated seed transport assembly including at least one holding member operable to transfer the singulated seeds from at least one elevator unit of the seed load assembly to the at least one sampling module of the seed sampling assembly. In connection therewith, the at least one sampling module includes a plurality of sampling positions each associated with a sampler, wherein the at least one sampling module is operable to remove a tissue sample from a seed at one of the sampling positions while another of the sampling positions is being cleaned to remove residual seed tissue therefrom.

Description

[0001] This application is a divisional application of the invention patent application filed on June 19, 2018, with application number 201880041785.3 and entitled "Automated System and Related Method for Removing Tissue Samples from Seeds".

[0002] Cross-reference to related applications

[0003] This application claims the benefit and priority of U.S. Provisional Application No. 62 / 523,072, filed June 21, 2017. The entire disclosure of the above application is incorporated herein by reference. Technical Field

[0004] This disclosure generally relates to automated systems and methods for removing tissue samples from biological materials, such as seeds. Background Technology

[0005] This section provides background information in relation to this disclosure, which is not necessarily prior art.

[0006] In plant development, genetic improvement is achieved through selective breeding or genetic manipulation, and when the desired improvement is realized, commercial production is developed or scaled up by planting and harvesting seeds over several generations. However, not all harvested seeds express the desired trait, and therefore it is necessary to weed out these seeds from a large quantity. To accelerate the process of increasing the seed quantity, a statistical sample can be taken from the original number of seeds and tested to weed out seeds that do not adequately express the desired trait (or seed groups associated with the statistical sample). Summary of the Invention

[0007] This section provides a general overview of this disclosure, but is not a full disclosure of the entire scope or all features of this disclosure.

[0008] Exemplary embodiments of this disclosure generally relate to automated seed sampling components. In one such embodiment, the automated seed sampling component typically includes at least one sampling module having a plurality of sampling locations, each associated with a sampler, wherein the at least one sampling module is operable to remove tissue samples from seeds at one of the sampling locations while cleaning another of the sampling locations to remove residual seed tissue therefrom.

[0009] Exemplary embodiments of this disclosure also generally relate to seed sampling systems. In one such embodiment, the seed sampling system typically includes an automated seed loading assembly operable to pick a single seed from a plurality of seeds (or load a single seed from a set of individually held seeds), wherein the seed loading assembly includes a plurality of laterally spaced lifting units, each of which is operable to actuate one of the picked seeds to a position generally above the lifting unit. The system also includes an automated seed sampling assembly including a plurality of laterally spaced sampling modules operable to remove a tissue sample from one of the picked seeds; and an automated seed delivery assembly including a plurality of laterally spaced holding members operable to transfer the picked seed from the lifting units of the seed loading assembly to the sampling modules of the seed sampling assembly. In this regard, the lateral spacing between the lifting units of the seed loading assembly, the lateral spacing between the sampling modules of the automated seed sampling assembly, and the lateral spacing between the holding members of the automated seed delivery assembly are generally the same or approximately the same.

[0010] Exemplary embodiments of this disclosure further relate generally to an automated method for removing tissue samples from seeds. In one such embodiment, a method typically includes picking a seed from a plurality of seeds; engaging the picked seed with a holding member of an automated seed delivery assembly; orienting the seed at the holding member to move the oriented seed to a sampling module of an automated seed sampling assembly; and removing a tissue sample from the picked seed at the sampling module.

[0011] Further areas of application will become apparent from the description provided herein. The descriptions and specific examples in this overview are intended for illustrative purposes only and are not intended to limit the scope of this disclosure. Attached Figure Description

[0012] The accompanying drawings described herein are for illustrative purposes only, for the purpose of selecting embodiments and not all possible implementations, and are not intended to limit the scope of this disclosure.

[0013] Figure 1 It is a perspective view of a seed sampling system that includes one or more aspects of this disclosure and is configured to pick seeds individually and remove tissue samples from the picked seeds.

[0014] Figure 2 yes Figure 1 Another perspective view of the seed sampling system;

[0015] Figure 3 yes Figure 1 Side view of the seed sampling system;

[0016] Figure 4 yes Figure 1 A perspective view of a portion of the seed load component of the system, the perspective view showing the queuing station of the seed load component;

[0017] Figure 5 yes Figure 1 A perspective view of another part of the seed load assembly of the system, the perspective view showing the seed picking unit of the seed load assembly;

[0018] Figure 6 yes Figure 5 A perspective view of a portion of a seed picking unit, the perspective view showing its hopper and separating wheel;

[0019] Figure 7 yes Figure 5 Another perspective view of a portion of the seed picking unit, which further shows its hopper and separating wheel;

[0020] Figure 8 yes Figure 1 A perspective view of a portion of the system's seed load component, along with the seed imaging component and the seed sampling component.

[0021] Figure 9 yes Figure 8 A partial view, which further shows a portion of the seed load assembly and the imaging assembly;

[0022] Figure 10 yes Figure 8 A partial perspective view of the lift unit of the seed load assembly;

[0023] Figure 11 yes Figure 1 A perspective view of the seed delivery components of the system;

[0024] Figure 12 yes Figure 1 A perspective view of a portion of the system's seed load component, along with the seed imaging component and the seed sampling component.

[0025] Figure 13 yes Figure 1 A partial perspective view of the seed sampling component of the system, from which the sampling module is disassembled;

[0026] Figure 14 yes Figure 1 A perspective view of an exemplary sampling module for seed sampling in a system;

[0027] Figure 15 yes Figure 14 A partial perspective view of the sampling module, showing the housing of the sampling module disassembled;

[0028] Figure 16 yes Figure 15 A magnified partial perspective view of the sampling module;

[0029] Figure 17 yes Figure 1 A perspective view of the sample collection components of the system;

[0030] Figure 18 yes Figure 17 A perspective view of the nozzle block of the sample collection assembly;

[0031] Figure 19 yes Figure 18 A partial cross-sectional view of the nozzle block;

[0032] Figure 20 yes Figure 1 A perspective view of the seed collection component of the system;

[0033] Figure 21A It is possible Figure 1 A perspective view of an exemplary implementation of a seed tray used in the system;

[0034] Figure 21B It is possible Figure 1 A perspective view of an exemplary embodiment of the sample plate used in the system;

[0035] Figure 22 yes Figure 1 A block diagram illustrating an exemplary relationship between a system and a control system suitable for or used therewith;

[0036] Figure 23 It is possible Figure 22 A block diagram of the computing device used in an exemplary arrangement.

[0037] Throughout the accompanying drawings, reference numerals indicate the corresponding parts in several views. Detailed Implementation

[0038] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. The descriptions and specific examples included herein are intended for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0039] Figure 1-20An exemplary embodiment of an automated seed sampling system 10, incorporating one or more aspects of this disclosure, is shown. The illustrated system 10 is suitable for removing samples from biological material (e.g., sampling the material, cutting the material, etc.). Samples may include, for example, tissue samples. Furthermore, biological material may include, for example, seeds. Again, the exemplary embodiments are provided for illustrative purposes only and may be used in conjunction with one or more methods disclosed herein.

[0040] like Figure 1-3 As shown, the seed sampling system 10 typically includes an automated seed loading assembly 12, an automated seed delivery assembly 14, an automated seed imaging assembly 16, and an automated seed sampling assembly 18. Generally, the seed loading assembly 12 operates (as part of the method herein) to individually pick (or separate, or select, etc.) individual seeds from a number (e.g., multiple, etc.) of seeds, and / or load a group of individual seeds (e.g., a group of such individually picked seeds, etc.) onto the seed sampling system 10. Furthermore, the seed delivery assembly 14, typically positioned above the seed imaging assembly 16 and the seed sampling assembly 18, operates to move the individually picked seeds from the seed loading assembly 12 to the seed imaging assembly 16, and then to the seed sampling assembly 18, wherein tissue samples (e.g., a single sample from each of the seeds, multiple samples from each of the seeds, etc.) are ultimately removed from the individually picked seeds. The tissue samples and the seeds from which the tissue samples have been removed are collected to maintain a relationship between them (e.g., a one-to-one relationship, so that the seeds can subsequently be identified based on the samples, etc., removed therefrom). The tissue sample can then be analyzed to determine whether the corresponding seeds from which the tissue sample was obtained exhibit one or more desired traits. Furthermore, based on the analysis, the corresponding seeds from which the tissue sample has been removed can subsequently be identified and used as needed.

[0041] The operation of the seed sampling system 10 and its seed loading assembly 12, seed delivery assembly 14, seed imaging assembly 16, and seed sampling assembly 18 is automated and can be controlled (and / or coordinated) for example, by a central control system (broadly, a computing device, etc.) within the scope of this disclosure. Furthermore, components of the seed loading assembly 12, seed delivery assembly 14, and / or seed sampling assembly 18 can be pneumatically operated, for example, by a desired airflow. Such pneumatic operation can be applied to move seeds through the seed sampling system 10 and between assemblies 12, 14, and 18. Such pneumatic operation may also include attracting seeds through the sampling system 10 (e.g., via vacuum, etc.), forcing seeds through the system 10 (e.g., via an air jet, etc.), actuating components of the seed sampling system 10, and / or combinations thereof, for example, to help suppress seed damage during delivery, to facilitate the efficient operation of components of the system 10, etc.

[0042] In the illustrated embodiment, the seed loading assembly 12, seed delivery assembly 14, seed imaging assembly 16, and seed sampling assembly 18 are supported by various structures such as mounting brackets, beams, platforms, bases, brackets, etc., and include various couplers (e.g., valves, pipe connectors, etc.). Although such structures and / or couplers are necessary for the construction of the seed sampling system 10, a description of the placement, orientation, and interconnection of such structures and / or couplers is not necessary for those skilled in the art to readily and fully understand the structure, function, and operation of the seed sampling system 10. In particular, such structures are clearly shown throughout the figures, and their placement, orientation, and interconnection are readily understood by those skilled in the art.

[0043] The seed loading component 12 of the seed sampling system 10 includes a queuing station 20 for receiving seeds from seed bags or other seed-containing devices (e.g., tubes, pools, boxes, cylinders, plates, etc.) for sampling. The seed bags may include any desired type and / or quantity of seeds, for example, as described herein. The seed bags may represent different items or groups of seeds that require analysis for one or more reasons (e.g., one or more reasons described herein, etc.). Each seed bag typically includes a marker associated with it (e.g., barcode, QR code, RFID tag, magnetic tag, magnetic stripe, alphanumeric marker, another marker, etc.). The marker can then be used to identify logical data regarding the corresponding seed bag (and the seeds contained therein). This logical data may be generated based on a specific genotype or attribute of each particular seed in the seed bag and may include, for example, characteristics and / or traits of the seeds, such as type, size, shape, color, composition, quality, weight, hereditary traits, etc. Additionally, the logical data may include data indicating whether to analyze the seeds in the seed package, the specific analysis to be performed for the seeds to be analyzed, and specific sampling requirements for the analysis of the seeds and / or their requirements (e.g., including a number of tissue samples to be obtained from the seeds, etc.). The logical data can then be used by the central control system (or directly by system 10) to set, guide, update, modify, etc., various components of system 10 as described herein, to remove appropriate tissue samples from a given tissue and to enable the performance of appropriate analyses of the tissue samples (particularly, for example, in the case where system 10 is integrated with one or more analysis units configured to perform the different analyses described herein). Accordingly, such logical data may relate to (but is not limited to) the type of seeds in the seed package, the sample size of such seeds, the analysis to be performed, the number of samples required for such analysis, etc. The logical data can be compiled in any suitable or desired format; for example, the logical data can be compiled into one or more electronic data structures, databases, spreadsheets, and / or lookup tables, etc., that are then accessible to the seed sampling system 10 (e.g., via a suitable network, etc.) and / or its users.

[0044] As an example, to initiate the operation of the seed sampling system 10, a tag from a given seed package can be input to the control system (e.g., via a user interface, via communication with a reader / input device, etc.), which communicates with the seed sampling system 10 via a network, etc. Specifically, for example, the queuing station 20 may include a reader configured to scan (broadly, read) the tags on a given seed package, or a separate reader (e.g., a handheld scanner input device, etc.) may be used to scan the tags. In either case, a processor associated with the control system can then access logical data associated with the seed package in a logical data structure (e.g., in a data structure in memory associated with the processor of the control system, in a remote data structure accessible via a network, etc.). Based on this logical data, the processor can then control the operation of the system 10 (even if the processor may not be explicitly mentioned) as described in detail below to set custom processing conditions (e.g., air pressure, vacuum pressure, component position, timing, tissue removal parameters, etc.) to remove the desired tissue sample from the seeds in the given seed package. In various implementations, the markings associated with the seed package can be automatically read or interpreted via a user interface and automatically input into the control system. In one example, the markings may include barcodes, and the user interface may include a suitable barcode reader. Thus, to initiate operation of system 10, a user or operator can use a barcode reader to scan the barcode, and the processor of the control system can then interpret the barcode, access logical data in a data structure corresponding to the barcode, and control the operation of system 10 as appropriate (e.g., based on the logical data, system 10 can determine the sample size, sample quantity, etc. of the seeds in the seed package; etc.).

[0045] For further reference Figure 4When scanning a given seed packet, and when the seed sampling system 10 is used to sample and analyze the corresponding seeds in the seed packet, the system 10 is configured to actuate the door 28 of the queuing station 20 (e.g., open door 28, unlock door 28, etc.) to allow one or more desired seeds from the seed packet to be received into the queuing station 20 (e.g., based on an initial scan, etc.). Relatedly, the queuing station 20 includes a filter unit 30 (e.g., a filter screen, a magnetic rod, a combination thereof, etc.) for removing unwanted and / or undesirable contaminants from the received seeds. As seeds move through the filter unit 30, they are received in one of a plurality of queues 32 of the queuing station 20, ready for subsequent processing. In the illustrated embodiment, queuing station 20 includes six queues 32, each separated by a movable barrier 34 (or door) for selectively holding (and isolating) different groups of seeds from different seed packets received in queuing station 20, so that the six different groups of seeds or items can be processed sequentially in the illustrated system 10 as needed (where each group is held in one of the six different queues 32). It should be understood that queuing station 20 may include other numbers of queues 32 in other embodiments (e.g., at least one, at least two, more than six, etc., in addition to six), depending on operational requirements, etc. Furthermore, queuing station 20 may be configured such that different queues in queue 32 can be processed together (e.g., seeds in different queues in queue 32 can be moved together to system 100, etc.) to potentially create larger queues (consisting of multiple queues in individual queues 32, etc.) to accommodate a larger number of seeds.

[0046] Then, in seed sampling system 10, when the required seeds (from the required number of seed packets) are received in queuing station 20, seed sampling system 10 is configured to allow one of the queues 32 (e.g., Figure 4 Seeds in the bottommost queue 32 (etc.) are moved to the seed picking unit 36 ​​of the seed loading assembly 12 (e.g., via induced airflow such as vacuum pressure and a suitable tube (not shown)).

[0047] Now for reference Figure 5-7 After the seeds are received at the seed picking unit 36 ​​(via inlet 38), the seed velocity is initially reduced by the seed decelerator 40. Figure 5 The process slows down / lowers, and the seeds are then collected in migration queue 42. Once all seeds from a given seed pack have been collected in migration queue 42, the seeds are then released (via automated gate 44) into hopper 46. Hopper 46 defines and includes a storage tank 47 for receiving and holding the seeds therein. Figure 6 and Figure 7(For example, all seeds from a given seed pack in a migration queue, etc.). The separating wheel 48 is then configured to communicate at least partially with the reservoir 47 of the hopper 46 (and specifically with the seeds in the reservoir 47). The separating wheel 48 is configured to rotate relative to the hopper 46 (via the motor 50). And, as... Figure 6 As best shown in the diagram (where cover 52 is detached from the separating wheel 48), the orifices 54 of the separating wheel 48 (in conjunction with a vacuum source) are configured to capture individual seeds from groups of seeds in the hopper 46 and retain the seeds in the orifices as needed (via a desired vacuum pressure, e.g., based on given logical data of the seeds, based on specific seeds received in the system (e.g., the vacuum pressure may be configured to a specific value based on seed type, seed size, seed quality, etc.)) and potentially optimize seed pickup efficiency. Sensors 56 are positioned near the separating wheel 48 to, for example, sense whether individual seeds are correctly captured in the respective orifices 54 (e.g., one seed in one orifice 54, etc.), count the seeds as they enter the orifices 54 and / or move through the sensors 56 (e.g., as part of quality control for monitoring the number of seeds entering and leaving the seed sampling system 10, etc.), their combinations, etc. In other exemplary embodiments, the seed sampling system may include a seed loading assembly with a separating wheel having a different number and / or size of orifices. Furthermore, in other exemplary embodiments, the seed sampling system may include a seed loading assembly with individual picking units that utilize features other than a separation wheel to individually pick seeds (e.g., a vibrating separator, etc.). For example, in other exemplary embodiments, the seed loading assembly of the seed sampling system may be configured to load one or more plates containing individual seeds onto or onto the system. Relatedly, the system may additionally include a queuing system (or a queuing feature associated with the seed loading assembly) with a motion actuator (e.g., an arm, etc.) that moves one or more desired seeds from the plates to a transfer tube connected to the seed loading assembly (thereby automating the loading of the seeds onto the system substantially via the queuing system, etc.).

[0048] In operation (and as part of the method of this disclosure), the separating wheel 48 of the seed picking unit 36 ​​rotates (via motor 50) to move the orifice 54 generally through the reservoir 47 of the hopper 46. As the separating wheel 48 rotates, suction is provided to the orifice 54 (via a vacuum source) such that the orifice 54 passing through and / or adjacent to the hopper reservoir 47 captures and holds individual seeds within the orifice 54. As the separating wheel 48 continues to rotate, it moves the orifice 54 and removes the captured seeds from the hopper reservoir 47 and generally moves them away from the hopper reservoir, past the sensor 56, and to the deposition chamber 58. In the deposition chamber 58, the captured seeds (via reduced suction within the orifice 54 and / or via a scraper (not shown)) are removed from the orifice 54 and received (e.g., via gravity, vacuum pressure, etc.) in a conveying chamber (not visible) extending to the deflector 60. The separating wheel 48 then continues to rotate and eventually moves the emptied orifice 54 back to the hopper reservoir 47 to capture additional seeds from the hopper 46 (where appropriate), for example until all seeds from a given seed bag in the hopper 46 have been transferred to the diverter 60, or until the desired number of seeds from the hopper 46 have been transferred to the diverter 60, etc.

[0049] In the illustrated embodiment, the hopper 46 of the seed picking unit 36 ​​includes a discharge gate 62. Figure 7 After a seed project is completed (i.e., after all required seeds have been picked from the seed project list), if any seeds remain in hopper 46 (and cannot be transferred to deflector 60 or are not intended to be transferred to deflector 60), system 10 is configured to actuate discharge gate 62 (e.g., open discharge gate 62, etc.) so that any remaining seeds in reservoir 47 of hopper 46 can be removed and collected in the desired disposal container (thus preparing hopper 46 to receive seeds from queuing station 20 for use in another seed package associated with another project). Relatedly, other features such as pressurized air may be used within hopper 46 to help ensure that any remaining seeds are removed from hopper 46 through discharge gate 62 and conveyed to the disposal container.

[0050] Special Reference Figure 5The deflector 60 of the seed sorting unit 36 ​​is typically positioned below the separating wheel 48 (and below the sedimentation chamber 58). The deflector 60 is configured to receive seeds removed from the separating wheel 48 and individually distribute each seed to the deflector manifold 66. Furthermore, the deflector 60 is configured to rotate between multiple different positions to align with one of a plurality of conduits 68 extending through the deflector manifold 66, thereby (e.g., via gravity, induced airflow, mechanical operation, etc.) transferring individual seeds from the hopper 46 to the appropriate conduit in the conduit 68 (e.g., thus defining multiple separate seed paths for seeds moving forward through the system 10). For example, when the deflector 60 transfers a single seed to one of the conduits 68, the deflector then rotates to align with another conduit 68 and transfers another single seed thereto. This process can be repeated until each of the conduits 68 in the manifold 66 receives a single seed. Relatedly, sensors (not shown) may be associated with the steering manifold 60 and / or conduit 68 to, for example, sense seeds received in the steering manifold 60 and / or conduit 68, count the seeds when they enter the steering manifold 60 and / or conduit 68, count the seeds when they leave the steering manifold 60 and / or conduit 68, combinations thereof, etc. In the seed sampling system 10 shown, the steering manifold 66 includes seven conduits 68 (although in Figure 5 (Only three are visible in the image). Furthermore, of the seven conduits 68, six are configured to guide seeds to the sampling assembly 18, and one is configured to guide seeds to a disposal container as needed or where appropriate (e.g., excess seeds received by the diverter 60, particularly seeds received by the diverter 60 based on sensor-acquired data). However, it should be understood that the diverter manifold 66 may include other numbers of conduits in other embodiments (e.g., at least one, at least six, at least seven, at least eight, etc.), for example, based on multiple seed paths to be defined by and / or included in the system 10 (and typically having at least one additional conduit for seed disposal as needed).

[0051] like Figure 8-10As shown, the seed loading assembly 12 also includes a plurality of lift units 70 (e.g., six lift units 70 in the illustrated embodiment) for receiving individually picked seeds from the steering manifold 66. The lift units 70 are generally positioned below the seed picking unit 36 ​​(and therefore generally below the steering 60 and the steering manifold 66). Each of the lift units 70 communicates with one of the conduits 68 of the steering manifold 66 (e.g., via a delivery pipe (not shown) extending from the conduit 68 to the inlet 72 of the lift unit 70). Thus, individually picked seeds from the manifold 66 can be transferred (e.g., via gravity, induced airflow, etc.) to the lift units 70 for subsequent transfer to the seed delivery assembly 14 (as part of a plurality of separate seed paths for individually picked seeds in system 10, i.e., where each lift unit constitutes part of each seed path)). Generally, when the lift unit 70 is empty and ready to receive seeds (e.g., when previous seeds at lift unit 70 have been transferred to seed delivery assembly 14, etc.), single-picked seeds are transferred from the diverter manifold 66 to lift unit 70. Relatedly, single-picked seeds can be transferred one at a time from diverter manifold 66 to lift unit 70 (e.g., when one of the conduits 68 of manifold 66 receives a seed from diverter 60, it can immediately transfer said seed to the corresponding one in lift unit 70, etc.). Alternatively, single-picked seeds can be held in diverter manifold 66 until all conduits 68 are filled with seeds, and then all seeds in conduits 68 are transferred sequentially or typically simultaneously to the corresponding lift units of lift unit 70.

[0052] like Figure 10 As specifically shown in the diagram (one example of an elevator unit 70 is illustrated), the elevator unit 70 includes a retracted position (such as...) Figure 10 A piston 74 is movable (e.g., via pneumatic operation, etc.) between a raised position (typically above the retracted position) and a lowered position. When in the raised position (or when in the retracted position), the piston 74 can receive seeds from the steering manifold 66 onto its end portion 76 (via inlet 72 and a corresponding channel (not visible) guiding from inlet 72 through the lift unit 70 to the piston 74). The piston 74 is then configured to present the seeds for transfer / handover to the seed delivery assembly 14 (for subsequent delivery to the seed imaging assembly 16 and the seed sampling assembly 18). In various embodiments, the end portion 76 of the piston 74 may include a suction cup (e.g., a vacuum suction cup as described below) for receiving and retaining the seeds (e.g., via suction from negative pressure applied to the piston 74, etc.). However, it is understood that this is not necessary in all embodiments of system 10.

[0053] Furthermore, in the lifting unit 70, the piston 74 can be actuated from the raised position to the retracted position (again, as...). Figure 10 As shown), in the retracted position, the end portion 76 of the piston 74 is exposed to the outlet 78. If the transfer to the seed delivery assembly 14 is missed, or if multiple seeds are detected in the lift unit 70 at a given time, or if seeds are detected (via sensors at the lift unit) to have one or more specific characteristics (e.g., undesirable characteristics based on intermediate analysis, specific size, specific type, etc.), the piston 74 can be actuated to the retracted position, for example, to discharge seeds from the lift unit 70 through the outlet 78 (e.g., via gravity, via compressed air source 80, via vacuum pressure, etc.) (e.g., to a residue bin, another location, etc.). Relatedly, sensors or other imaging devices may be associated with the lift unit 70 to sense seeds received from the manifold 66, to count seeds as they enter the lift unit, to evaluate seeds to be discharged from the lift unit 70 (e.g., to evaluate specific characteristics of the seeds, etc.), and / or combinations thereof (e.g., as the last point or opportunity to remove or discharge seeds from the system 10 in the seed path before sampling and processing the seeds, and thereby affecting the collection operation of the system 10; etc.). Furthermore, after the seeds have been successfully transferred to the seed delivery assembly 14 (e.g., via compressed air source 80, etc.), the piston 74 may be actuated to a retracted position to substantially clean the lift unit 70, for example, when determined by one of the sensors to be necessary.

[0054] Accordingly, it should be understood that the separation wheel 48 and the steering mechanism 60 of the seed picking unit 36, combined with the conduit 68 of the steering manifold 66, allow for the picking of individual seeds from the quantity initially received in the hopper 46 (in relation to a given seed package). Thus, the seed loading assembly 12 operates to provide individual seeds to the seed delivery assembly 14, which is then transferred to the seed imaging assembly 16 and the seed sampling assembly 18 (so that individual seed identification is typically recorded and tracked in the system 10 from this point as part of the individual seed path through the system 10). Furthermore, and as described above, sensors configured in communication with one or more of the steering mechanism 60, the steering manifold 66 (and its conduit 68), and / or the lifting unit 70 help to further ensure that only one seed is transferred from the seed loading assembly 12 at a time (thus contributing to the single seed identification characteristic of the system 10). Furthermore, additional seed-related data, including, for example, infrared (IR) images, near-infrared (NIR) images, seed color, seed size, disease grading, etc., can be captured via sensors and / or imaging components / units described herein (which may be located (unlimited) at the separating wheel 48, steering gear 60, and lifting unit 70, and may additionally include other sensors and / or imaging components / units described herein), via sensors and / or imaging components / units described herein. Such data can then be used by system 10 to enhance upstream and / or downstream operations (e.g., sampling setup, process flow rate, etc.) and / or to remove or discharge specific seeds from the system based on one or more related classifications or other methods (e.g., via discarding conduit 68 at manifold 66, via outlet 78 at lifting unit 70, etc.) for disposal, sorting, collection, etc.

[0055] Now for reference Figure 11 The seed delivery assembly 14 of the seed sampling system 10 typically includes a translation mechanism 82 and a plurality of retaining members 84 (e.g., six retaining members 84 in the illustrated embodiment) mounted in a conveyor 86 supported by the translation mechanism 82. The illustrated translation mechanism 82 typically includes a first bracket 88 coupled to a guide 90, whereby the first bracket 88 is movable in the overall linear direction of the guide 90 (e.g., sliding via an actuator, via a motor drive unit, etc.). The translation mechanism 82 also includes a second bracket 92 coupled to a drive 94 (e.g., coupled to a belt drive, chain drive, etc.), whereby the second bracket 92 is movable in the overall linear direction via movement of the drive 94 (typically perpendicular to the movement of the first bracket 88). In this way, the translation mechanism 82 is configured to move the conveyor 86 and retaining members 84 relative to the seed load assembly 12 (and particularly relative to its lifting unit 70) in two directions. For example, the seed delivery assembly 14 is typically positioned above the lift unit 70 of the seed load assembly 12, and also above the seed imaging assembly 16 and the seed sampling assembly 18 (see also...). Figure 3Relatedly, the first carriage 88 is configured to move the conveyor 86 generally horizontally within the system 10 (in a generally parallel direction aligned with the elevator unit 70, the imaging unit 96 of the seed imaging assembly, and the sampling module 98 of the seed sampling assembly 18, e.g., as shown in the image). Figure 1 As shown, in the X direction of system 10, etc., and generally moving vertically (e.g., as...). Figure 1 As shown, in the Z direction of the system, etc.

[0056] The holding member 84 of the seed delivery assembly 14 may extend from the delivery unit 86 (e.g., via piston 100, etc.) and is configured to move at an angle as needed. This allows the holding member 84 to move as needed to retrieve (and capture) seeds from the lifting unit 70 (e.g., even when the raised seeds are not immediately aligned vertically with the holding member 84, etc.). Furthermore, the holding member 84 is also configured to rotate so that once the seeds are retrieved from the lifting unit 70, the holding member 84 is operable to orient the seeds in a desired orientation, position, etc. Relatedly, the holding member 84 includes an end portion 102 configured to retain, hold, etc., seeds received from the lifting unit 70. In the illustrated embodiment, the end portion 102 includes a suction cup (e.g., a vacuum suction cup, etc.) for receiving and retaining seeds (e.g., via negative pressure suction, etc.). The suction cup may include a cup-shaped end portion defining, for example, a V-shape, U-shape, or other shape conducive to holding the seeds. The suction cups are configured such that when negative pressure is applied to the suction cups (in a suitable manner), seeds can be engaged and thus retained (one seed is received in one of the suction cups). Then, as the seed is effectively transferred to the sampling assembly 18 via the holding member 84 and its end portion 102 releases the seed, positive pressure can be applied to the suction cups (at the end portion 102) (again, in a suitable manner) to generally clean the end portion 102 and help suppress any buildup and help improve seed pickup efficiency. In other exemplary embodiments, the seed sampling system may include a seed delivery assembly with a holding member, the end portion of which defines, in addition to the suction cups for receiving and retaining the seed, a mechanical holder, seed clamping mechanism, etc.

[0057] In the operation of the seed delivery assembly 14 (when the lifting unit 70 of the seed loading assembly 12 moves the seeds to an elevated position), the first bracket 88 is configured to generally position the conveyor 86 above the lifting unit 70, and the second bracket 92 is then configured to move the retaining member 84 to a position just above its piston 74 (so that each of the retaining members 84 is above the corresponding one in the lifting unit 70). Furthermore, the retaining member 84 (specifically, the end portion 102 of the retaining member 84) is configured to then engage the lifting unit 70 and receive the seeds from said lifting unit. As described above, this may involve actuating the retaining member 84 as needed to allow its end portion 102 to properly engage the seeds (e.g., extending the retaining member 84 relative to the conveyor 86 toward the seeds, moving the retaining member 84 at an angle relative to the conveyor 86, etc.). Furthermore, once the seed is engaged (and captured), the second bracket 92 of the seed delivery assembly 14 is configured to raise the delivery unit 86 (and the captured seed), and the first bracket 88 is configured to move the seed to the seed imaging assembly 16, as described below.

[0058] The seed imaging component 16 of the seed sampling system 10 is in Figure 12 The image is shown and is constructed and operable to image each of the seeds captured by the seed delivery assembly 14. Specifically, the seed imaging assembly 16 is configured to collect at least one image of each of the seeds held in the holding member 84 of the seed delivery assembly 14 (when the seed delivery assembly 14 moves the seed to the seed imaging assembly 16). The images of the seeds collected at the seed imaging assembly 16 can be any desired type of image. For example, the images can be visual images (color and / or black and white), IR images (correlated with IR bands) (e.g., to “view” haploid seeds, etc.), NIR images, or NMR / MRI images, or any other type of image or associated spectral data. Furthermore, the images can include two-dimensional images (through which two-dimensional (2-D) seed measurements of each of the seeds can then be collected, including (but not limited to) cap / tip position, seed area, seed shape, disease, etc.), or the images can include three-dimensional (3-D) images obtained from multiple 2-D images, or 3-D measurements can be obtained using a laser profilometer or any combination of techniques.

[0059] Once the images are collected, they are transmitted to the control system for storage in a relevant data structure and processing as described herein. For example, the images can be used to determine the seed orientation at the holding member 84 and to guide the operation of the holding member 84 to rotate and orient the seed in the desired position prior to a sampling operation. Relatedly, for example, the images can be used to position the seed embryo so that the seed can be oriented (by the holding member) in the desired position, thereby allowing the sample to be removed from the seed without damaging the embryo when the seed is delivered to the sampling assembly 18. Furthermore, for example, when performing a sampling operation, such as for single-seed phenotypic analysis, the images can be used to assist in the analysis of the seed in relation to any tissue analysis performed on the tissue sample removed from the seed (e.g., to determine seed volume and / or seed shape, identify diseases, identify non-viable seed material, etc.) and / or as part of a quality control procedure to monitor the operation of the seed sampling system 10 (e.g., to help adjust (e.g., speed up, slow down, etc.) various processes of system 10 (e.g., the processes of seed loading assembly 12, seed delivery assembly 14, seed sampling assembly 18, etc.) without interrupting the processes, etc.). Furthermore, for example, the image can be used to guide the operation of the seed sampling component 18 in removing tissue from the seed (e.g., directly operating the seed sampling component 18, etc.).

[0060] In the illustrated embodiment, the seed imaging assembly 16 includes a plurality of imaging units 96, which are generally positioned below the lift unit 70 and generally positioned between the lift unit 70 and the sampling module 98 of the seed sampling assembly 18 (see also...). Figure 8 The imaging unit 96 is generally aligned with the opening 104 in the base plate 106 of the seed sampling system 10 to allow access to seeds held at the holding member 84 of the seed delivery assembly 14 via the imaging unit 96. Accordingly, the imaging unit 96 may include, for example, a camera capable of capturing images of the type described above (and / or suitable for a particular imaging application of the system 10). Additionally, in some embodiments, the seed imaging assembly 16 may also include (e.g., as part of or in combination with the imaging unit 96) one or more light sources configured to illuminate the field of view of the imaging unit 96 as needed (although such light sources are not required in all embodiments). When present, the one or more light sources may include any type of light source suitable for a particular imaging application of the system 10 (e.g., incandescent lamps, fluorescent lamps, ultraviolet lamps, infrared (IR) lamps, light-emitting diodes (LEDs), etc.). Accordingly, the illustrated system 10 includes three imaging units 96, wherein each imaging unit is configured to image seeds associated with two adjacent seed paths of the system 10. However, it should be understood that in other embodiments, system 10 may include other numbers of imaging units (e.g., depending on the number of seed paths in system 10, etc.), and / or system 10 may include one imaging unit for each seed path.

[0061] In operation of the seed imaging assembly 16, the first carriage 88 of the seed delivery assembly 14 is configured to move the conveyor 86 (and the captured seeds) from the lift unit 70 to a position above the seed imaging assembly 16 (in the X direction of the system 10), such that the field of view (through the opening 104) of each of the imaging units 96 includes at least the bottom portion of at least one seed captured in the seed delivery assembly 14 (and more specifically, in the illustrated embodiment, two adjacent seeds, such that the two adjacent seeds are within the field of view of each of the imaging units 96, wherein each imaging unit 96 then captures one or more images of the two seeds). The second carriage 92 of the seed delivery assembly 14 is then configured to lower the conveyor 86 and the seeds toward the imaging units 96 (in the Z direction of the system 10), wherein the imaging units 96 capture one or more images of the seeds. In various embodiments, the second bracket 92 may be configured to lower the conveyor 86 such that the seed moves through the opening 104 of the base plate 106 and into a position adjacent to the imaging unit 96 (so that the imaging unit 96 is configured to collect images of multiple portions of the seed, such as when the seed is lowered (thus collecting an image of the bottom portion of the seed) and after the seed is positioned adjacent to the imaging unit 96 (thus collecting images of the side portions of the seed). Once the desired images have been collected, the seed delivery assembly 14 is configured to raise the seed (via the second bracket 92) and move the seed (via the first bracket 88) to the seed sampling assembly 18 (again in the X direction of the system 10). In other embodiments, the seed delivery assembly 14 may simply move the captured seed from the lift unit 70 to a position above the seed imaging assembly 16 (in the X direction of the system 10), whereby the imaging unit 96 then captures one or more images of the seed as described above (and lowers the seed toward the imaging unit 96 even without the seed delivery assembly 14).

[0062] Then, based on image data of the seed collected at the seed imaging assembly 16 (e.g., as evaluated by a control system), the holding member 84 is configured to rotate the seed to a desired orientation before presenting it to the seed sampling assembly 18 for sampling. Specifically, for example, in the illustrated embodiment, the seed can be oriented by the holding member 84 to avoid the embryo during sampling operations, thereby maintaining seed viability. Alternatively, in various other embodiments, the seed can be oriented to actually target the embryo or a specific part of the seed during sampling operations. In any case, the seed can be oriented to a desired orientation based on desired or detectable genotype, natural or non-natural traits, phenotype, etc. (including, but not limited to, seed oil content, moisture content, color, geometry, geometric classification such as flat or round, or process outcome, etc.). As an example, the seed can be oriented by the holding member 84 such that the seed cap or a particular side is ultimately presented to the sampling assembly 16 for sampling (e.g., to its sampler 114, etc.).

[0063] refer to Figure 13-16 The seed sampling component 18 of the seed sampling system 10 includes a plurality of sampling modules 98 (e.g., six sampling modules 98 in the illustrated embodiment, etc.). Furthermore, each of the sampling modules 98 includes two sampling locations 108, 110 for removing tissue (for performing the sampling operation) from the seed as it is presented to the sampling module 98 via the seed delivery component 14. In this way, each of the sampling modules 98 can accommodate parallel sampling and cleaning operations (potentially contributing to the throughput of the system 10), i.e., for each of the sampling modules 98, a seed can be sampled at a first sampling location 108 of the sampling module 98 while cleaning a second sampling location 110 (e.g., approximately at the same time, etc.), as described in more detail below. Additionally, each of the sampling modules 98 is configured via a calibration process to determine the relative position of the holding member 84 of the seed delivery component 14 to help facilitate precise transfer of the seed from the holding member 84 to the effective sampling locations 108, 110 of the sampling module 98 (which will be described in more detail below). Although the illustrated embodiment includes six sampling modules 98, it should be understood that embodiments of system 10 may include any desired number of sampling modules (e.g., at least one, at least six, six or more, etc.) within the scope of this disclosure, whereby the number of sampling modules may generally correspond to the number of seed paths in / through system 10, etc.

[0064] Special Reference Figure 14-16The following description will focus on one of the sampling modules 98; it should be understood that the descriptions of the other sampling modules 98 are substantially the same. The sampling module 98 shown typically includes a central seed holding assembly 112 configured to hold a seed in one of sampling positions 108, 110 (depending on which of the sampling positions 108, 110 is valid for sampling); and a sampler 114 for removing tissue from the seed held at a particular location in sampling positions 108, 110 (as part of the sampling operation of the sampling module 98). Relatedly, at each of the sampling positions 108, 110, the seed holding assembly 112 includes a pair of generally opposing arms 116 and a corresponding pad 118 for securing / holding the seed therebetween. An actuator 120 (e.g., a pneumatic clamp, etc.) is provided to move each of the corresponding arms 116 and the corresponding pads 118 bidirectionally toward and away from each other, thereby facilitating the fixation / holding (and subsequent release) of the seeds. In some embodiments, the two pairs of arms 116 of the seed holding assembly 112 (at the two sampling positions 108, 110) can move together (so that the two pairs of arms 116 are open or closed); while in other embodiments, the arms 116 of the seed holding assembly at the first sampling position 108 can move independently of the arms 116 at the second sampling position 110. Furthermore, in some embodiments, the pads 118 of the seed holding assembly 112 can be detached from the arms 116 to allow replacement pads to be installed on the arms 116 and / or to allow different pads to be installed on the arms 116 to accommodate different types of seeds, etc.

[0065] The seed clamping component 112 of the sampling module 98 can also be located within the sampling module 98. Figure 15 In the direction indicated by arrow 121 (e.g., typically in the X direction of system 10, etc.), via actuator 122 (see... Figure 12 (e.g., via a stepper motor, etc.) Thus, when a seed is held between a pair of arms 116 of the seed holding assembly 112, the seed holding assembly 112 enables the seed to move toward a sampler 114 associated with a particular sampling position 108, 110 for sampling operations. This allows the sampling module 98 to control the sampling feed rate of the seed toward the corresponding sampler 114 (based on the movement of the seed holding assembly 112 (e.g., speed, etc.) and the sampling depth of the tissue removed from the seed (based on the distance moved by the seed holding assembly 112). It should be understood that these characteristics can be controlled independently for each of the sampling modules 98 in the seed sampling assembly 18 (and for each of the samplers 114 at the different sampling positions 108, 110 at each of the sampling modules 98), thereby adapting the sampling operation in the system 10 to each sampling module 98 and each seed.

[0066] As shown above, the sampling module 98 includes two samplers 114, one of which is located at each of the sampling positions 108, 110 (for removing tissue from the seed held in the clamping assembly 112 at a respective sampling position 108, 110). In the illustrated embodiment, each of the samplers 114 includes a drill (e.g., a high-speed drill with controllable rotation per minute, etc.) and an associated drill bit (e.g., in the illustrated embodiment, the two drill bits are oriented along a common longitudinal axis). In some embodiments, each of the samplers 114 can be configured for different types of seeds and / or for removing different types and / or sizes of tissue samples from the seeds. For example, tissue sample sizes as low as 4.5 mg can be achieved (e.g., depending on the seed type, depending on sample analysis requirements, etc.). Accordingly, in other embodiments, the sampling module 98 may include other samplers (in addition to the drill and drill bit) for removing tissue from the seed, including, for example, a cutting wheel, broach, knife, laser, etc. Furthermore, in some embodiments, the sampling module 98 may include different types of samplers at each of the sampling positions 108, 110 (e.g., a drill at the first sampling position 108 and a cutting wheel at the second sampling position 110, etc.) and / or include different types of samplers at each of the sampling modules 98.

[0067] like Figure 15 and 16 As shown, the sampling module 98 also includes a first sensor 124 and a second sensor 126 at each of the sampling positions 108, 110. As will be described below in conjunction with the operation of the seed sampler assembly 18, the sensors 124, 126 help facilitate, control, monitor, etc., the reception of seeds from the seed delivery assembly 14 to the sampling module 98, and the movement of the seed holding assembly 112 relative to the sampler 114 at each of the sampling positions 108, 110 (depending on which of the sampling positions 108, 110 is valid for sampling) in relation to the sampling operation of the sampling module 98.

[0068] Specifically, for example (and generally as described above), each of the sensors 124, 126 of the sampling module 98 is configured via a calibration process to determine the relative position of the seed holding assembly 112 (and its arm 116) and the holding member 84 of the seed delivery assembly 14 to help facilitate precise transfer of the seed from a given holding member 84 to a selected sampling position 108, 110 of the sampling module 98. Furthermore, once the seed is positioned in the seed holding assembly 112, each of the sensors 124, 126 is configured via a calibration process to further determine the relative position of the seed holding assembly 112 (and the seed held therein) and a corresponding one of the samplers 114 to facilitate precise removal of tissue from the seed. Thus (and possibly further based on image data collected for a given seed at the seed imaging component 16), the specific type of the sampled seed can be identified (thereby the system 10 can adapt to different types of seeds and control the operation of the clamping component 112 and the sampler 114 to adapt to a specific type of seed where appropriate), and tissue samples of the desired size and / or shape can be removed from the seed by selecting the sampler 114. Therefore, it should be understood that the seed sampling system 10 can be adapted to different types of seeds and / or adjust the size / shape of tissue samples by independently controlling each sampler 114 in each sampling module 98 or by consistently controlling any two or more samplers 114 (e.g., by adjusting the rotation per minute (RPM) of the sampler 114, by changing the RPM of the sampler 114 during actual sampling operations, by modifying the rate at which seeds are fed to the sampler 114, etc.) and / or by modifying / adjusting the position of the seeds held by a given seed holding assembly 112 (e.g., where the seeds are located between arms 116, etc.) and / or by modifying / adjusting the clamping pressure of the seeds applied to the seed holding assembly 112 by arms 116, etc.

[0069] In the operation of the seed sampling assembly 18, after the seed image data held in the seed delivery assembly 14 is collected by the seed imaging assembly 16 and after the seed is oriented (or at approximately the same time or before), the seed delivery assembly 14 is configured to move the seed to the seed sampling assembly 18 (again, in the X direction of the system 10). In doing so, a first bracket 88 is configured to position the conveyor 86 over the sampling module 98, and a second bracket 92 is configured to lower the conveyor 86 (and the holding member 84) to position the seed into the sampling module 98 (e.g., through a corresponding opening 128 in the housing 130 of the sampling module 98). Specifically, the seed delivery assembly 14 is configured to position the seeds at specific sampling positions (i.e., sampling positions 108, 110 that are effective for the sampling operation) within the sampling positions 108, 110 of the sampling module 98, and at a height that typically corresponds to the arms 116 and / or the sampler 114 (as determined by one or more of the sensors 124, 126, etc.) via the corresponding openings in the openings 128. Then, the first sensor 124 of the seed holding assembly 112 checks, determines, and identifies, for example, the outer extensions of the seeds (e.g., the actuator 120 of a given seed holding assembly 112, etc.), and based on this, the seed holding assembly 112 is configured to move as needed to position the seeds at desired locations between their arms 116 (and corresponding pads 118) (e.g., moving the seed holding assembly 112 from a starting position to a seed capture position, etc.). For example, if a seed is oriented to the cap position via the seed delivery assembly 14, the first sensor 124 can then position the seed cap such that the gripping assembly 112 holds the seed relative to the gripping surface of the liner 118 in the desired position and orientation (e.g., where the seed cap protrudes from the gripper liner 118 (e.g., about one millimeter, etc.)). The seed gripping assemblies 112 are configured to then actuate their arms 116 together to hold the seed between them. Furthermore, the holding member 84 is configured to release the seed (e.g., terminate any negative pressure suction applied thereto, etc.), and the seed delivery assembly 14 returns to the lifter unit 70 to capture additional seeds. Again, it should be understood that image data collected by the seed imaging assembly 16 (and / or by any other imaging and / or sensing device herein) can be used at the seed sampling assembly 18 (e.g., in combination with sensors 124, 126; etc.) to help position the seed individually at the correct height between the arms 116 of the seed holding assembly 112, thereby controlling the exact location of tissue removal of the seed (and possibly determining the seed position prior to transfer to the holding assembly 112 and determining the position of the sampler 114).

[0070] For further reference Figure 15-17The exemplary sampling module 98 shown, when a seed is positioned at a first sampling position 108 between the arms 116 in the clamping assembly 112, establishes a negative pressure (e.g., vacuum pressure, etc.) in the sample collection funnel 132 to prepare for sampling, and the clamping assembly 112 moves the seed toward the corresponding sampler 114. In doing so, a second sensor 126 identifies the leading edge of the seed and captures the position of the seed edge relative to the sampler 114 (e.g., based on the movement of the clamping assembly 112 and the calibration position of the sampler 114 and the clamping assembly 112, etc.). In conjunction with this, the clamping assembly 112 moves toward the sampler 114 until the desired sample depth of the seed is reached (and possibly the desired amount, size, etc., of tissue is removed). In other embodiments, the sampler 114 may alternatively (or additionally) move toward the seed held in the clamping assembly 112 until the desired sample depth of the seed is reached. For example, sampler 114 may be generally movable within sampling module 98 in the X direction of system 10 via an actuator (such as actuator 122) (e.g., via a stepper motor, etc.). Furthermore, removed tissue is drawn into sample collection funnel 132 via a negative pressure airflow. Clamping assembly 112 then moves back to its initial position, and arm 116 releases seeds into seed collection funnel 134 via opening 136 (see...). Figure 13 As described above, each of the sampling modules 98 includes a corresponding component for facilitating sampling operations at each of the sampling positions 108, 110. Thus, each of the sampling positions 108, 110 of the sampling modules includes a similar sample collection funnel 132 and seed collection funnel 134 (and corresponding openings 136) operable in the manner described above. Accordingly, in various embodiments, the system 10 may also include one or more sensors and / or imaging components / devices associated with collecting removed tissue from the seeds (e.g., downstream of seed collection funnel 123, etc., when the removed tissue is drawn into seed collection funnel 134) and configured to measure and / or otherwise quantify the amount of tissue removed from the seeds. In this way, such data can provide control input to the depth setting of the sampler 114 and clamping assembly 112 during the sampling operation to help ensure that an accurate amount of tissue is removed from a given seed.

[0071] In the illustrated embodiment (and as described above), the sampling module 98 of the seed sampling assembly 18 is configured to remove tissue from the seed in a non-destructive manner so as to preserve the seed's germination potential. This is described in more detail below.

[0072] Now for reference Figure 17-20Tissue removed from the seeds at sampling module 98 is captured (via sample collection funnel 132) and conveyed (e.g., via gravity, air pressure, air jet, etc.) to sample collection assembly 138 of seed sampling system 10. Similarly, seeds with removed tissue are captured (via seed collection funnel 134) and conveyed (e.g., via gravity, air pressure, air jet, etc.) to seed collection assembly 140 of seed sampling system 10. Relatedly, tissue samples are collected at sample collection assembly 138 in sample plate 142 (e.g., in specific holes of plate 142), and seeds are collected at seed collection assembly 140 in seed trays (not shown) (e.g., in specific holes of seed trays), such that a known relationship exists between each of a particular seed and the tissue removed therefrom. For example, one or more identifiers may be assigned to the seeds and / or the tissue samples removed therefrom. Thus, the seeds and the tissue samples obtained from the seeds can subsequently be associated. Furthermore, various data (e.g., various image data, etc.) captured by system 10 for a given seed, as well as subsequent tissue analysis data, can be associated with the appropriate seed within the seed, for example, at the control system, via an identifier. Accordingly, and as will be understood from the following description, both sample collection assembly 138 and seed collection assembly 140 include corresponding sample collection components and seed collection components for each of the sample locations 108, 110 in each sampling module 98 of system 10. In this way, tissue removed from the seed at the sampling module 98, along with the corresponding seed, can be collected, while maintaining a single seed identity (including the identity of the corresponding sample removed from the seed) within system 10.

[0073] In particular and as Figure 17-19As shown, the sample collection assembly 138 includes a sample plate platform 144 adapted to securely retain a sample plate 142 in a fixed position and orientation, and two nozzle blocks 146 generally located above the sample plate platform 144 and configured to transfer tissue removed from the seed at the sampling module 98 to the sample plate 142. Each sample plate 142 includes a plurality of orifices, each of which is adapted to receive a tissue sample extracted from the seed by one of the sampling modules 98 (via a corresponding one of the nozzle blocks 146). The nozzle blocks 146 include a plurality of discharge nozzles 148, each of which is in fluid communication with one of the sampling positions 108, 110 of the sampling module 98 (via a tube 150 extending from the sample collection funnel 132 to the corresponding discharge nozzle of the discharge nozzle 148). Thus, each of the sampling positions 108, 110 of the sampling module 98 includes a dedicated path to an orifice in one of the sample plates 142 at the sample collection assembly 138. In the illustrated embodiment, each nozzle block 146 includes six discharge nozzles 148, totaling twelve discharge nozzles 148 between the two nozzle blocks 146, which is equal to the total number of sampling positions 108, 110 at the sampling module 98 in the seed sampling assembly 18. Furthermore, the discharge nozzles 148 are spaced apart and arranged to generally correspond to the spacing and arrangement of the holes within the sample plate 142.

[0074] Furthermore, the sample plate platform 144 of the sample collection assembly 138 is mounted to an XY stage 152, which includes an X-axis translation rail 154 and a Y-axis translation rail 156. An actuator is then operated to move the sample plate platform 144 bidirectionally relative to the nozzle blocks 146 along the lengths of the X-axis translation rail 154 and the Y-axis translation rail 156 to a desired position (e.g., via one or more devices similar to a actuator 94). Additionally, each nozzle block 146 is mounted to a linear actuator 158 (e.g., a pneumatic slider), operable to move a corresponding nozzle block 146 bidirectionally in the Z-direction of the system 10 (e.g., up and down relative to the sample plate platform 144, etc.). Thus, the sample plate platform 144 enables the orifice of the sample plate 142 to be moved in the XY direction of the system 10 to a specific position below the nozzle blocks 146 (e.g., to target a position below the nozzle blocks 146, etc.). Furthermore, the nozzle block 146 can then be moved in the Z direction of the system 10 to deposit the tissue sample removed from the seed at a sampling module 98 in a specific hole in the sample plate 142 (where the sample plate 142 then receives the tissue sample).

[0075] Relatedly, in the operation of the sample collection assembly 138, before the sampling module 98 removes tissue from the seeds therein (as described above), the sample collection assembly 138 operates to move the orifices of the sample plate 142 in the XY direction of the system 10 (via the sample plate platform 144 and the XY stage 152) to a specific location below the nozzle block 146 (e.g., to target a location below the nozzle block 146, etc.). The nozzle block 146 is then configured to move in the Z direction of the system 10 to lower the discharge nozzle 148 and position the discharge nozzle to align with a corresponding orifice in the orifice of the sample plate 142. In the illustrated embodiment, each of the discharge nozzles 148 is configured to contact and seal with a corresponding one of the orifices (e.g., via an O-ring, gasket, bushing, etc.). This helps ensure that substantially all tissue discharged from the discharge nozzle 148 is deposited into the corresponding orifice without cross-contamination from adjacent samples escaping around the discharge nozzle 148. Furthermore, as described above, the discharge nozzles 148 are spaced apart and arranged to generally correspond to the spacing and arrangement of the holes within the sample plate 142. Thus, when the nozzle blocks 146 are lowered, the six discharge nozzles 148 of each of the nozzle blocks 146 are configured to contact and seal with one of the holes in the sample plate 142 (so that tissue samples removed from different seeds at different sampling modules 98 can potentially be deposited into different holes in the sample plate 142 through one of the nozzle blocks 146 at a given time).

[0076] Then, for each of the sampling modules 98, when the tissue sample is actually removed from the seed (as described above), the tissue is drawn into the corresponding sample collection funnel 132 and conveyed through the tube 150 to the corresponding nozzle block 146 (which again extends from the given sample collection funnel 132 at the particular sampling module 98 to the corresponding discharge nozzle 148 at the nozzle block 146). The tissue is then deposited through the discharge nozzle 148 into a corresponding hole in the sample plate 142 (whereby each of the tissue samples from the six different sampling modules 98 is directed to a different hole in the sample plate 142). As part of this operation, the tissue is drawn through the tube 150 via an induced airflow, where, for a given discharge nozzle 148, the air is then discharged through an adjusted exhaust port 160 at the nozzle block 146, while the tissue material remains in the flow path to be received in the specific hole. Once tissue has been received from each sampling module 98 in the wells of sample plate 142 (for a given sampling operation or sampling run), nozzle block 146 is configured to rise and sample collection assembly 138 is configured to position subsequent wells of sample plate 142 in target locations, whereby nozzle block 146 then descends again to prepare for delivery of additional tissue samples to sample plate 142 (for subsequent sampling operations or sampling runs via seed sampling assembly 18). In other embodiments, tissue samples may be obtained multiple times from a single seed, and each tissue sample may be aspirated into (and / or collected in) more than one well of sample plate. In doing so, system 10 may, for example, be used to separate external seed tissue (maternal) from internal seed tissue so that further genotyping can be targeted at the tissue source location of the seed. In even further embodiments, tissue samples from more than one seed may be aspirated into (and / or collected in) a single well of sample plate.

[0077] Furthermore, in system 10, imaging component 161 (e.g., "imaging camera", "laser profilometer", etc.) is associated with sample collection component 138 and is typically positioned between nozzle blocks 146 to collect image data of sample plate 142 (see [link to system 10]). Figure 17This image data can then be used to determine the presence of tissue within the wells of sample plate 142, and can also be used to quantify the amount, volume, or weight of tissue, and even further to determine the presence of contaminated tissue within the one or more wells prior to the sampling operation (and before receiving tissue samples into the one or more wells). The image data (and other image data captured by system 10) can also be used by the central control system, for example, to adjust seed sampling assembly 18, to help optimize tissue removal and provide adjustments to upstream / downstream processes (e.g., sorting operations, extraction of dilution targets, genotyping treatment, breeding submission requirements, selection decisions, etc.). Additionally, downstream genotyping detection data can be used in conjunction with the image data to determine the level of contamination. Furthermore, sensor 163 can be associated with nozzle block 146 (e.g., positioned adjacent to discharge nozzle 148, etc.) and configured to provide information on tissue measurement and / or quantification of tissue dispensed through nozzle block 146 (e.g., through each of discharge nozzles 148, etc.). Sensor 163 may include, for example (but not limited to), mass flow measurement sensors, such as optical through-hole sensors, microwave sensors, or other Doppler effect-based sensors.

[0078] Accordingly, it should be understood that, in various embodiments, the sampling operations performed by system 10 require specific timing for the aforementioned different operations in order to suppress contamination. In this regard, pressure sensors can be used to drive the processing timing herein (in addition to the various image data collected in system 10) to help ensure that the different components of system 10 are in the appropriate positions at the appropriate times.

[0079] Subsequently, the tissue samples received in sample plate 142 can be used to test and analyze various traits of the corresponding seeds from which the tissue samples have been removed (as described in more detail below).

[0080] In the illustrated embodiment, each nozzle block 146 of the sample collection assembly 138 includes an ionization rod 161 mounted to its underside (see [reference]). Figure 18 The ionization rod 161 is configured to help suppress static buildup on the nozzle block 146 and on the sample plate platform 144 and / or sample plate 142. Furthermore, the tube 150 of the sample collection assembly 138 may be made of a static dissipative material so that a portion of the tissue removed from the seed and delivered to the sample plate 142 does not adhere to the interior portion of the tube 150 and cause cross-contamination of the sample.

[0081] Figure 20Seed collection assembly 140 of seed sampling system 10 is shown. As shown, seed collection assembly 140 includes a seed tray platform 162 adapted to securely hold a seed tray (not shown) in a fixed position and orientation thereon; and a seed deposition unit 164 for guiding seeds to the seed tray. Each of the seed trays includes a plurality of seed wells, each of the seed wells adapted to receive a seed after the corresponding seed has been sampled by one of the sampling modules 98. For example, in various embodiments, each seed tray may be a twenty-four-well tray, etc. Accordingly, seed collection assembly 140 is configured to receive seeds from sampling module 98 of seed sampling assembly 18 in the wells of the seed tray in such a way that the seeds can subsequently be identified as specific tissue samples from which they have been removed.

[0082] The seed tray platform 162 of the seed collection assembly 140 is mounted to an XY stage 166, which includes an X-axis translation track 168 and a Y-axis translation track 170. An actuator is then operable to move the seed tray platform 162 bidirectionally relative to the seed deposition unit 164 along the X-axis translation track 168 and the Y-axis translation track 170 to a desired position (e.g., via one or more actuators similar to actuator 94). Furthermore, the seed deposition unit 164 is mounted to a linear actuator 172 (e.g., a pneumatic slider), operable to move the seed deposition unit 164 bidirectionally in the Z-direction of the system 10 (e.g., up and down movement). Thus, the seed tray platform 162 (via the XY stage 166) enables the holes of the seed tray to move in the XY direction of the system 10 to a specific position below the seed deposition unit 164 (e.g., to target a position below the seed deposition unit 164). Furthermore, the seed deposition unit 164 can then be moved in the Z direction of the system 10 to move the seed nozzle 174 to the appropriate position to deposit the seeds released / received from the sampling module 98 into specific holes in the seed tray (so that the seeds are received in the seed tray). Sensor 176 (in...) Figure 20A single seed (identified by only one seed) is then positioned at the seed deposition unit 164 to count the number of seeds passing through (e.g., to detect no seeds passing through, a single seed passing through, multiple seeds passing through, fragments passing through, etc.). In some embodiments, the seed tray platform 162 may also include an imaging component (e.g., including one or more imaging devices described herein), configured to determine whether a seed has been successfully received within the seed tray and whether a single seed has been captured in a given well of the seed tray; and / or configured to capture additional seed data such as seed size. Again, this data can be used by a central control system, for example, to adjust the seed sampling component 18, to help optimize tissue removal and to provide adjustments to upstream / downstream processes (e.g., sorting operations, extraction and dilution of targets, genotyping, breeding submission requirements, selection decisions, etc.).

[0083] Figure 21A An exemplary seed tray 167 is shown for use with the seed collection assembly 140, thereby allowing multiple seed trays in the seed tray 167 to be positioned on the seed tray platform 162. Relatedly, the illustrated seed tray 167 includes multiple compartments or holes 169 for receiving seeds from the nozzle block 146. Furthermore, Figure 21B An exemplary embodiment of a sample plate 171 (e.g., as an alternative to or part of sample plate 142) is shown for use with sample collection assembly 138 (e.g., positioned on sample plate platform 144, etc.). Relatedly, the illustrated sample plate 171 includes a plurality of compartments or holes 173. It should be understood that the sample plate 171 may have a configuration similar to that of the seed tray 167, and / or the number and arrangement of holes 169 in the seed tray 167 may correspond to the number and arrangement of holes 173 in the sample plate 171 (although this is not required in all embodiments). This correspondence facilitates a one-to-one correspondence between seeds and their samples. However, in some embodiments, it may be desirable to provide multiple compartments in the sample plate 171 (or sample plate 142) for each compartment in the seed tray 167, for example, where multiple tests can be performed on the sample, or where different samples can be obtained from the same seed (e.g., samples from different depths, etc.).

[0084] Furthermore, in system 10, imaging component 165 (e.g., imaging camera, laser profilometer, etc.) (see...) Figure 3Image data of the seed collection assembly 140, typically positioned above the seed tray platform 162, is associated with and generally positioned above the seed tray (and the seeds received in the holes therein). This image data can then be used, for example, to determine the presence of seeds in the holes of the seed tray, and can also be used to quantify the received seeds, their volume or weight, etc., and can even be further used to identify missed seed collections or seeds received in incorrect holes. The image data (and other image data captured by system 10) can also be used by the central control system, for example, to adjust the seed sampling assembly 18, to help optimize tissue removal, to provide adjustments to upstream / downstream processes (e.g., sorting operations, extraction and dilution of targets, genotyping, breeding submission requirements, selection decisions, etc.).

[0085] In the operation of the seed collection assembly 140, exactly before, simultaneously with, or immediately after the sampling module 98 removes tissue from the seeds therein (as described above), the seed collection assembly 140 operates to position the orifices of the seed plate at a specific location below (and relative to) the seed deposition unit 164. The seed deposition unit 164 is then configured to move in the Z direction of the system 10 to lower the seed nozzle 174 and position it aligned with a corresponding orifice in the orifice of the seed plate. Then, for each of the sampling modules 98, after the tissue sample is removed from the seeds, the seed holding assembly 112 is commanded to release the seeds into the corresponding seed collection funnel 134. The seeds are then guided via a suitable tube (not shown) extending from the seed collection funnel 134 to the corresponding seed nozzle 174 at the seed deposition unit 164 (e.g., via gravity, via induced airflow, etc.), where the seeds are then deposited into a specific orifice in one of the seed trays. Once a seed is received from each sampling module 98 in the holes of the seed tray, the seed deposition unit 164 is configured to rise, and the seed collection assembly 140 is configured to position the subsequent holes of the seed tray at target locations below the seed deposition unit 164, whereby the seed deposition unit 164 then descends again to prepare for the delivery of additional seeds to the seed tray.

[0086] Refer again Figure 17The sample collection assembly 138 of the seed sampling system 10 also includes two purge blocks 178 configured to be used in conjunction with the operation of the seed sampling assembly 18 to clean the flow path of tissue samples from the sampling module 98 (e.g., from sampling positions 108, 110 of the sampling module 98 through tube 150) to the nozzle block 146. Each of the purge blocks 178 is associated with one of the nozzle blocks 146 of the sample collection assembly 138. Thus, for each of the sampling modules 98, once the tissue removed from each seed is received in one or more sample plates 142 and the corresponding seed is received in a seed tray, the nozzle block 146 operates to lower (as described above) and seal against the purge block 178. Subsequently, the blow-out nozzle 180 of the sampling module 98 (combined with a negative pressure airflow at the sample collection funnel 132) is activated to force any residual seed tissue from the sampling module 98 to its associated vacuum collection section (or port) (i.e., to the collection funnel 132), which then guides the residual seed tissue to the nozzle block 178 (via tube 150) and purge block 178 for filtration and disposal. Furthermore, the seed path tube 150 extending from the sample collection funnel 132 to the corresponding discharge nozzle in the discharge nozzle 148 is also cleaned via an induced airflow therein, which is then filtered and disposed of (along with the residual seed tissue) at the purge block 178. Accordingly, it should be understood that all surfaces of the system 10 exposed to tissue are actively cleaned during targeted removal. All tissue is removed and filtered via a dedicated flow path.

[0087] Again, it should be understood that, in various embodiments, the cleaning operation performed by system 10 also requires specific timing of the aforementioned different characteristics in order to suppress contamination and ensure proper cleaning. Relatedly, and as stated above (and related to the above), pressure sensors can be used to drive the processing timing herein to help ensure that the different components of system 10 are in the appropriate positions at the appropriate times.

[0088] As described above, each of the sampling modules 98 is capable of accommodating parallel sampling and cleaning operations. Thus, while cleaning the first sampling position 108 of each of the sampling modules 98 (in the manner described above), the second sampling position of each of the sampling modules 98 can be used for seed sampling (and vice versa). The material selection for each of the sampling modules 98 (and its components) includes materials configured to mitigate contaminant accumulation and to adequately remove residual tissue to prevent contamination of downstream genotyping detection. Again, this feature of the seed sampling system can potentially contribute to the throughput of system 10.

[0089] In various embodiments, when sample plates 142 are positioned on the sample plate platform 144 of the sample collection assembly 138, the tray identification number (e.g., barcode, etc.) of each of the plates 142 and the position of the plate 142 on the platform 144 are recorded (as part of a given identifier for each of the tissue samples). Additionally, when each tissue sample is received into a well of the sample plate 142, the specific XY position of the well (and thus the sample) can be recorded. The recorded well positions on the sample plate 142 and the sample plate platform 144 can then be compared with the XY position of each deposited tissue sample to map the specific sample in each well of each sample plate 142. Similarly, when seed trays are placed on the seed tray platform 162 of the seed collection assembly 140, the tray identification number (e.g., barcode, etc.) of each seed tray and the position of each seed tray on the seed tray platform 162 are recorded (again, as part of a given identifier for each of the seeds). Additionally, since each seed is deposited in a well, the XY position of the well on the seed tray platform 162 can be recorded. The recorded well locations on the tray and seed tray platform 162 can then be compared with the XY location of each deposited seed to map the specific seed in each well of each seed tray. In this way, the seeds received in the seed tray can be associated with the tissue received in the sample plate 142.

[0090] In the illustrated embodiment, the sampling modules 98 of the seed sampling assembly 18 are typically designed to minimize tooling connections. Relatedly, each of the sampling modules 98 is detachable from the seed sampling assembly 18 and can be replaced by another sampling module 98, for example, to provide hardware-specific changes for a particular seed project, minimizing downtime during maintenance of a given sampling module 98, etc. Furthermore, each of the sampling modules 98 is configured for quick insertion into and removal from the seed sampling system 10. For example, as... Figure 13 and 14 As shown, each of the sampling modules 98 includes a plug 184 configured to be quickly inserted into or removed from the container 186 of the seed sampling assembly 18 of the system 10 (e.g., to provide power to the sampling module 98, etc.). Furthermore, the sampling module 98 can be quickly coupled to (and disconnected from) the seed sampling system 10 via couplers 190-194 of the seed sampling assembly 18 (the couplers corresponding to mating couplers (not shown) of the sampling module 98).

[0091] Furthermore, in the illustrated embodiment, the seed picking assembly 12 is shown to include six conduits 68 associated with the diverter manifold 66 and six lifter units 70; and the seed delivery assembly 14 is shown to include six holding members 84; the seed sampling assembly is shown to include six sampling modules 98; and the sample collection assembly is shown to include six pairs of discharge nozzles 148. However, it should be understood that in other embodiments, different numbers of these components of the seed sampling system 10 may be provided to adjust the throughput as needed. Additionally, the positioning of one or more components of the seed sampling system 10 may be modified to adjust the throughput of the system 10. Accordingly, in various embodiments, the system 10 may be configured to provide a sample throughput of approximately 7 seconds every 6 seed cycles (from seed entry into the system 10 to collection of the sample removed from the seeds and the sampled seeds).

[0092] Furthermore, in the illustrated embodiment and as described above, individual seed identities are typically recorded and tracked in the seed sampling system 10 from the seed picking assembly 12 to the sample collection assembly 138 and seed collection assembly 140. This is achieved at least in part by maintaining a separate seed path for each of the picked seeds from the lift unit 70 of the seed picking assembly 12 to the seed imaging assembly 16, to the seed sampling assembly 18, and to the sample collection assembly 138 and seed collection assembly 140 (via the seed delivery assembly 14). Relatedly, to facilitate such separate seed paths, the corresponding lift units in the lift unit 70 of the seed picking assembly 12, the imaging device 96 of the seed imaging assembly 16, and the sampling module 98 of the seed sampling assembly 18 are generally aligned in the X direction of the seed sampling system 10. In particular, the lateral spacing (in the Y direction of the seed sampling assembly 10) between the lift units 70 of the seed loading assembly, between the sampling modules 98 of the seed sampling assembly 18, and between the holding members 84 of the seed delivery assembly 14 is the same (or approximately the same).

[0093] Furthermore, in the illustrated embodiment, different groups of seeds associated with different seed packages can be migrated through the seed load component 12, one group at a time (via a separate seed path), thereby maintaining sample integrity through the sampling process (e.g., via the use of movable doors, barriers, etc.).

[0094] As described above, the seed sampling system (e.g., system 10, etc.) and method / operation of this disclosure are operable to protect, preserve, etc., the germination potential of sampled seeds, and are therefore, for example, considered non-destructive. For example, the size, location, and / or shape of the removed tissue sample can be precisely controlled to protect the germination potential of the sampled seeds. Germination potential represents the dominant number of sampled seeds that remain viable after sampling (i.e., greater than about 50% of all sampled seeds). In one particular embodiment, at least about 75% of the sampled seeds and in some embodiments, at least about 95% of the sampled seeds remain viable. It should be noted that in some cases or for certain applications, a lower rate of germination potential may be tolerable, for example, as genotyping costs decrease over time because a larger number of seeds can be sampled for the same genotyping cost. It should also be noted that sampling does not need to have any impact on viability at all.

[0095] In one embodiment, the germination potential of the sampled seeds is maintained for at least approximately six months after sampling to ensure that the sampled seeds will be viable until they reach the field for planting. In a particular embodiment, the sampled seeds undergo further treatment to maintain germination potential. Such treatment may generally include any means known in the art for protecting seeds from environmental conditions during storage or transport. For example, in one embodiment, the sampled seeds may be protected by storing them before planting or by treating them with polymers and / or fungicides while transporting them to the field.

[0096] The seed sampling system disclosed herein (e.g., system 10, etc.) defines a generally compact footprint. This compact footprint is allowed by the configuration of the system's seed loading assembly, seed delivery assembly, seed imaging assembly, and / or seed sampling assembly. The compact footprint (and compact dimensions) allow the system to be transported for operation at various locations.

[0097] The seed sampling system disclosed herein (e.g., system 10, etc.) is configured to accommodate different types of seeds and / or different sizes of seeds. For example, the orifice of the separating wheel can be configured to accommodate individual seeds among different types and / or sizes (e.g., automatically adjusting the variability of seed size via a brush, etc.) so that the sampling system can be used to process different types of seeds without changing the separating wheel. Furthermore, the end portion of the retaining member can be configured to retain individual seeds among different types and / or sizes. Additionally, the sampler (and associated sampling module) can be configured to sample individual seeds among different types and / or sizes of seeds.

[0098] Exemplary seeds that can be used with the seed sampling system (e.g., System 10, etc.) and method of this disclosure include alfalfa seeds, apple seeds, banana seeds, barley seeds, bean seeds, broccoli seeds, cabbage seeds, rapeseed seeds, carrot seeds, castor bean seeds, cauliflower seeds, Chinese cabbage seeds, citrus seeds, clover seeds, coconut seeds, coffee seeds, maize (or corn) seeds, cotton seeds, cucumber seeds, Douglas fir seeds, dried bean seeds, eggplant seeds, eucalyptus seeds, fennel seeds, green bean seeds, gourd seeds, and leek seeds. Seeds include: lettuce seeds, slash pine seeds, flax seeds, melon seeds, oat seeds, okra seeds, olive seeds, onion seeds, palm seeds, pea seeds, peanut seeds, pepper seeds, poplar seeds, pumpkin seeds, radish pine seeds, radish seeds, rapeseed seeds, rice seeds, rye seeds, spinach seeds, sorghum seeds, squash seeds, southern pine seeds, soybean seeds, strawberry seeds, beet seeds, sugarcane seeds, sunflower seeds, sweet corn seeds, sweetgum seeds, tea seeds, tobacco seeds, tomato seeds, turf seeds, watermelon seeds, wheat seeds, and Arabidopsis thaliana seeds. Furthermore, crops analyzed using seed and / or tissue samples obtained as disclosed herein may include forage crops, oil crops, cereal crops, fruit crops, ornamental plants, vegetable crops, fiber crops, spice crops, nut crops, turf crops, sugar crops, beverage crops, tuber crops, root crops, and forest crops.

[0099] Seeds and / or tissue samples obtained from said seeds using the seed sampling system of this disclosure (e.g., System 10, etc.) and related methods can be analyzed as needed. For example, the sampled seeds and / or their tissue samples can be analyzed for desired target traits (e.g., physical, chemical, morphological, and / or genetic characteristics; markers; genotypes, etc.). Generally, such traits are determined by analyzing the sample for one or more characteristics indicating at least one genetic or chemical trait. Furthermore, the analysis may include analyses for determining starch content, protein content, oil content, fatty acid distribution, etc.

[0100] Seeds and / or tissue samples obtained from said seeds using the seed sampling system of this disclosure (e.g., System 10, etc.) and related methods can also be used to facilitate germplasm improvement activities. For example, seeds and / or their tissue samples can be analyzed to identify and select seeds containing one or more desired traits (including natural or non-natural traits), markers, haplotypes, and genotypes. On the one hand, analytical methods can be included in conjunction with the seed sampling system of this disclosure (e.g., System 10, etc.) and related methods to allow for the analysis of individual seeds present in a batch or large batch of seeds, enabling the determination of the chemical and / or genetic characteristics of individual seeds.

[0101] Non-limiting examples of target traits include color (e.g., white versus red), size, shape, seed type, resistance to pests (e.g., insects, mites, fungi, yeasts, molds, bacteria, nematodes, weeds, and parasitic and saprophytic plants), falling numerical score (e.g., Hagberg value), baking or pasta quality, etc.

[0102] More specifically, non-limiting examples of characteristics indicating chemical properties include proteins, oils, carbohydrates, fatty acids, amino acids, biopolymers, pharmaceuticals, starch, fermentable starch, secondary compounds, metabolites, etc. Therefore, non-limiting examples of chemical properties include amino acid content, protein content, protein composition, starch content, fermentation yield, fermentation efficiency, energy yield, oil content, determination of protein profiles, determination of fatty acid distribution, determination of metabolite distribution, etc.

[0103] In addition, non-restrictive examples of characteristics indicating hereditary traits may include, for example, genetic markers, single nucleotide polymorphisms, simple sequence repeats, restriction fragment length polymorphisms, haplotypes, tag SNPs, alleles of genetic markers, genes, DNA-derived sequences, RNA-derived sequences, promoters, 5' untranslated regions of genes, 3' untranslated regions of genes, microRNAs, siRNAs, quantitative trait loci (QTLs), satellite markers, transgenes, mRNA, ds mRNA, transcriptomic profiles, methylation patterns, ploidy (or levels), etc.

[0104] In one embodiment, the seed sampling system (e.g., System 10, etc.) and related methods of this disclosure can be used to remove tissue samples from wheat seeds. Any desired characteristics of the tissue sample can then be analyzed (e.g., color (e.g., white vs. red, etc.), protein composition, falling number fraction, baking or noodle quality, etc.). Based on this analysis (e.g., based on the presence of one or more desired characteristics, etc.), the sampled wheat seeds can be selected for further use (e.g., further analysis, cultivation, packaging, use in breeding operations, etc.).

[0105] In one implementation, seed samples obtained using a seed sampling system (e.g., System 10, etc.) and related methods include endosperm tissue capable of determining allele frequencies, thereby making it possible to infer parental linkage phases for specific markers. Furthermore, comparisons of allele frequency data between two or more germplasm banks provide insight into target selection, thereby inferring that increases in allele frequencies are associated with changes in the distribution of one or more traits in conjunction with said one or more target traits. Additionally, evaluation of relative allele frequency data between lines can contribute to the construction of genetic linkage maps.

[0106] In another embodiment, seed samples obtained using a seed sampling system (e.g., System 10, etc.) and related methods can be used with double haploid technology to aid germplasm improvement activities, including reducing the double haploid procedure by selecting only preferred seeds for doubling. For example, seed samples can be obtained comprising haploid and double haploid materials, and the genotypic and chemical characteristics of the seed samples can be analyzed. The seed samples can then be used for trait integration and evaluation, as well as marker-assisted breeding.

[0107] Seed and / or tissue samples obtained from seeds using the seed sampling system of this disclosure (e.g., System 10, etc.) and related methods can also be used in breeding programs to select plants or seeds with desired genetic or chemical traits, wherein desired genetic traits include genotype, haplotype, alleles, sequence, transcriptome profile, and methylation pattern. For example, seed and / or tissue samples can be used in combination with any breeding method and can be used to select a single generation or multiple generations. The choice of breeding method depends on the manner in which the plant reproduces, the heritability of the trait is to be improved, and the type of cultivar used commercially (e.g., F1 hybrid cultivar, pure-line cultivar, etc.). Non-limiting methods for selecting breeding plants are listed below. It should also be understood that any commercial and non-commercial cultivar can be used in breeding programs. Factors including, but not limited to, germination vigor, nutrient vigor, stress tolerance, disease resistance, branching, flowering, fruit setting, seed size, seed density, erectness, and threshing ability will generally determine the selection.

[0108] In one particular implementation, seeds and / or tissue samples obtained from said seeds using seed sampling systems (e.g., System 10, etc.) and related methods are used to determine the genetic characteristics of the seeds in a marker-assisted breeding program. This allows for improved marker-assisted breeding programs where direct seed sampling (as disclosed herein) can be performed while maintaining the identity of individual seeds from the seed sampling system (e.g., System 10, etc.) to the field. As a result, marker-assisted breeding programs produce a “high-throughput” and more efficient platform where seed populations with the desired traits, markers, or genotypes can be accumulated more efficiently in shorter time periods, requiring less field and labor resources. Such advantages will be described more fully below.

[0109] In some exemplary embodiments, seeds and / or tissue samples obtained from said seeds using the seed sampling system of this disclosure (e.g., System 10, etc.) and related methods can be used in conjunction with methods for analyzing the presence or absence of at least one genetic marker in nucleic acids extracted from said seeds and / or samples. The desired seeds can then be selected based on the results of the nucleic acid analysis, for example, for cultivating plants. Relatedly, System 10 can be integrated with a corresponding tissue analysis unit, whereby tissue samples removed from the seeds can be automatically delivered to the analysis unit (e.g., sample plates can be delivered to the analysis unit independently of human intervention, etc.).

[0110] For example, DNA can be extracted from tissue samples using any DNA extraction method known to those skilled in the art, which will provide sufficient DNA yield, DNA quality, PCR reaction, and sequencing method reaction. A non-limiting example of a suitable DNA extraction method is SDS-based extraction using centrifugation. Furthermore, the extracted DNA can be amplified post-extraction using any amplification method known to those skilled in the art. For example, a suitable amplification method is from Amersham Biosciences. DNA amplification preparation.

[0111] In addition (or alternatively), RNA can be extracted from tissue samples using any RNA extraction method known to those skilled in the art, which will provide sufficient RNA yield, RNA quality, PCR reaction, and sequencing method reaction. A non-limiting example of a suitable RNA extraction method is SDS-based extraction using centrifugation, considering reagents and materials that are free of RNases. Furthermore, the extracted RNA can be amplified post-extraction using any amplification method known to those skilled in the art. For example, a suitable amplification method is Full Spectrum from System Biosciences. TM RNA amplification.

[0112] The presence or absence of appropriate genetic polymorphisms in the extracted nucleic acids is analyzed. A wide variety of genetic markers used to analyze genetic polymorphisms are available and known to those skilled in the art. As used herein, genetic markers include, but are not limited to, simple sequence repeats (SSRs), single nucleotide polymorphisms (SNPs), insertions or deletions (Indels), single characteristic polymorphisms (SFPs) or transcriptomic profiles, and nucleic acid sequences. Nucleic acid analysis targeting the presence or absence of genetic markers can be used to select seeds in breeding populations. This analysis can be used to select genes, QTLs, alleles, or genomic regions (haplotypes) that contain or are linked to genetic markers. In this document, analytical methods are known in the art and include, but are not limited to, PCR-based detection methods (e.g., TaqMan assays), microarray methods, and nucleic acid sequencing methods. Novel molecular biology techniques combined with modifications to classical breeding strategies can be used to identify genes, alleles, QTLs, or haplotypes to be selected.

[0113] In one of these exemplary embodiments, the sampled seeds are selected based on the presence or absence of one or more traits genetically linked to the QTL. Examples of QTLs of general interest include, but are not limited to, herbicide tolerance, disease resistance, insect or pest resistance, altered fatty acid, protein or carbohydrate metabolism, increased grain yield, increased oil content, increased nutrient content, increased growth rate, enhanced stress tolerance, preferred maturity, enhanced sensory properties, altered morphological characteristics, other agronomic traits, traits for industrial use, traits that enhance consumer appeal, and combinations of traits as multi-trait indices. Alternatively, seeds may be selected based on the presence or absence of one or more traits genetically linked to the haplotype associated with the QTL. Examples of such QTLs may again include, but are not limited to, herbicide tolerance, disease resistance, insect or pest resistance, altered fatty acid, protein or carbohydrate metabolism, increased grain yield, increased oil, increased nutrient content, increased growth rate, enhanced stress tolerance, preferred maturity, enhanced sensory properties, altered morphological characteristics, other agronomic traits, traits for industrial use, traits that enhance consumer appeal, and combinations of traits as multi-trait indices.

[0114] If homozygous inbred parents are used in the initial breeding cross, breeding population selection can begin as early as the F2 breeding level. If one or more parents in the cross are heterozygous for the target allele or marker, sampling and advancement can also be performed in the F1 generation. Breeders can analyze the F2 population to retrieve the marker genotype for each individual in that population. The initial population size (limited only by the number of seeds available for analysis) can be adjusted to meet the required probability of successfully identifying the desired number of individuals. Therefore, the probability of finding the desired genotype, the initial population size, and the target population size can be modified for various breeding methods and the level of inbreeding in the sampled population.

[0115] Depending on the breeding method and target, the selected seeds can be stockpiled or stored separately. For example, when a breeder is analyzing the disease resistance of an F2 population, all individuals with the desired genotype can be stockpiled and planted in a breeding nursery. Conversely, if multiple QTLs with different effects on traits such as grain yield are to be selected from a given population, the breeder can retain individual identities and go to the field for individuals with various combinations of the target QTL.

[0116] Several methods for preserving a single seed identity may be used when transferring sampled seeds from the sampling location (e.g., from seed sampling system 10, etc.) to the field. Methods include, but are not limited to, transferring selected individuals (e.g., directly from seed sampling system 10, etc.) to trays (e.g., seed trays, etc.), seed strips, box trays, index trays, or transplanting the sampled seeds together with peat moss pots and artificially planting them from individual seed packets, or directly labeling individual seeds with numbers, letters or alphanumeric characters, or barcodes (e.g., via inkjet printing or laser engraving, etc.).

[0117] Depending on the breeding target and genetic complexity, multiple selection cycles can be utilized.

[0118] The advantages of using the seed sampling system (e.g., System 10, etc.) and related methods (including analytical methods and seed breeding methods) of this disclosure include, but are not limited to, reducing the labor and field resources required per population or breeding line, improving the ability to evaluate larger breeding populations per field unit, and improving the ability to analyze desired traits in breeding populations before planting. Field resources per population are reduced by limiting the field space required to advance the desired genotype. For example, a population of 1,000 individuals planted with 25 seeds per row would consume a total of 40 rows in a field. Using conventional tissue sampling, all 1,000 plants would be labeled, and sampling would be done manually by scoring leaf tissue. Molecular marker results would be required before pollination, and pollination would only be performed on those plants containing the desired genetic makeup. Thus, if 50 seeds are determined to contain the desired genetic makeup, conventional breeding methods would require planting 1,000 plants to retain the desired 50 seeds. In contrast, this disclosure allows breeders to analyze 1,000 seeds in a laboratory and select the 50 desired seeds before planting. Fifty individuals can then be planted in the field, consuming only two rows of 25 seeds each. Furthermore, this disclosure allows breeders to avoid marking or sampling in the field, thus significantly reducing the required manual labor resources.

[0119] In addition to reducing the number of field rows per population, the seed sampling system (e.g., System 10, etc.) and related methods (including analytical methods and seed breeding methods) of this disclosure further allow for an increase in the number of populations that a breeder can evaluate in a given breeding nursery. Using the above example, where 50 seeds in a population of 1000 seeds contain the desired genetic makeup, a breeder applying the techniques of this disclosure can evaluate 20 populations of 50 seeds each using conventional field tissue sampling techniques with the same field area consumed by a single population. Even when selecting populations for a single allele using a 1:2:1 expected segregation ratio for F2 populations, a breeder can evaluate four populations in the same field area as a single field tissue sampled population.

[0120] A potential further advantage of using the seed sampling system (e.g., System 10, etc.) and related methods (including analytical methods and seed breeding methods) of this disclosure is the mitigation of risks associated with growing plants in certain geographic areas where plants may grow poorly or experience adverse environmental conditions, or even be destroyed in storms. For example, seeds with an “optimal” genotype or marker composition can be planted in geographic area 1, and seeds with a “second-best” genotype can be planted in geographic area 2. In this case, geographic area 2 serves as a backup in case any problems arise with the plants growing in geographic area 1. This is very difficult to do with conventional methods of obtaining tissue samples from germinating plants for genotyping, as these plants would subsequently need to be uprooted and transplanted to the second geographic area. Using the seed sampling system (e.g., System 10, etc.) and related methods (including analytical methods and seed breeding methods) of this disclosure avoids the problems of transplantation and also simplifies the logic of the breeding procedure.

[0121] In some embodiments, the seed sampling system (e.g., System 10, etc.) and related methods (including analytical methods and seed breeding methods) of this disclosure can be further used in breeding procedures to infuse traits into plants. Here, the presence or absence of at least one genetic marker in nucleic acids extracted from tissue samples is analyzed. Seeds are then selected based on the results of the nucleic acid analysis, and plants are cultivated from the selected seeds. The cultivated plants can then be used as female parents or male parents for hybridization with other plants.

[0122] Examples of genetic analysis for trait integration in seed selection include, but are not limited to, identifying high recurrence of parental allele frequencies, tracking target transgenes or screening for unwanted transgenes, selecting seeds for hybridization testing, selecting seeds expressing target genes, selecting seeds expressing heritable phenotypes, identifying seeds with selected genetic loci, and conjugation testing.

[0123] Using the seed sampling system (e.g., System 10, etc.) and related methods (including analytical methods and seed breeding methods) of this disclosure, high recurrence of allele frequencies again allows for a reduction in the number of rows per population and an increase in the number of planted populations or inbred lines in a given field unit. Therefore, this disclosure can also effectively reduce the resources required to complete the transformation of inbred lines.

[0124] The seed sampling system (e.g., System 10, etc.) and related methods of this disclosure, along with the tissue samples obtained therefrom (and the described analytical methods and seed breeding methods), further provide quality assurance (QA) and quality control (QC) by ensuring the identification and discarding of regulated or unwanted transgenes, undesirable genetic traits, or undesirable genetic phenotypes prior to planting. This application of QA capability effectively eliminates unintended release violations. A further extension of this disclosure is the screening for the presence of infectious agents and the removal of contaminated seeds prior to shipment.

[0125] The seed sampling system (e.g., System 10, etc.) and related methods (as well as the described analytical and seed breeding methods) disclosed herein can be further applied to identify hybrid seeds for transgenic testing. For example, in the transformation of inbred lines at the BCnF1 stage, breeders can efficiently create hybrid seed batches (excluding gamete selection) that are 50% hemizygous for the target trait and 50% homozygous for the lacking trait to generate hybrid seeds for testing. Breeders can then analyze all F1 seeds produced in the test cross and identify and select those that are hemizygous. The advantage of this method is that inferences drawn from hybridization experiments will represent commercial hybridization genetics regarding trait conjugation.

[0126] In one instance, the systems and methods of this disclosure can be used to evaluate segregation aberrations in transgenic seeds. Seeds from an F1 cross between line A (homozygous events 1 and 2) and line B (homozygous event 1) were induced in maternal haploid-induced segregation. The resulting seeds were selected using embryo color to obtain a population of presumed haploid seeds.

[0127] Individual putative haploid grains can be selected from the putative haploid seed population and sampled non-destructively using an automated seed sampler system (e.g., seed sampling system 10 as generally described herein). Markers are applied to the samples to determine the presence of Event 2 and Event 1 genes. The sampling process removes some pericarp and endosperm tissue, which is used as the basis for analysis. It is important to note that the endosperm tissue is triploid and contains genetic contributions from both parents. If the target gene is detected using this method, the presence of the desired gene in the haploid embryo can be accurately predicted. For the purposes of this study, samples from 180 grains were analyzed, and data were obtained for 175 grains due to sampling issues. Relatedly (and as described above), system 10 enables embryo-targeted sampling / tissue removal that generates true double haploid genetic information without the presence of the inducing genome (triploid nature).

[0128] As shown in Table 1, if each pair of Event 1 genes in the expected seed samples is positive and approximately 50% of the seed samples are positive for Event 2 genes, then the absence of segregational aberration is confirmed.

[0129] Table 1

[0130]

[0131]

[0132] The results of this study indicate that high-throughput, non-destructive seed sampling can be used as a screening mechanism to select individual genetic traits based on haploids.

[0133] Other applications of the seed sampling system (e.g., System 10, etc.) and related methods (including the described analytical methods and seed breeding methods) disclosed herein include the identification, tracking, and superposition of target traits, which have the same advantages as those identified above regarding required field and labor resources. Generally, transgenic conversion programs are performed in multi-seasonal locations, leading to higher land and management cost structures. Therefore, compared to temperate applications, the impact of reducing row requirements per population or increasing the population size within a given field unit is more significant on a cost basis.

[0134] The seed sampling system (e.g., System 10, etc.) and related methods (including the described analytical methods and seed breeding methods) disclosed herein can also be used from seeds of plants having two or more transgenes, wherein the accumulation or superposition of transgenic regions into a plant or strain is achieved by adding transgenes via transformation or by hybridization of parental plants or strains containing different transgenic regions, or any combination thereof. Analysis can be performed to select individual seeds based on the presence of one or more characteristics associated with at least one transgene. Such characteristics include, but are not limited to, the transgene itself, genetic markers linked to the transgene, mRNA expressed from the transgene, and protein products of the transgene.

[0135] Furthermore, the seed sampling system (e.g., System 10, etc.) and related methods (including the described analytical and seed breeding methods) of this disclosure can be used to improve the efficiency of double haploid procedures by selecting desired genotypes and identifying ploidy levels at the haploid stage to eliminate the processing and advancement of non-haploid seeds to the field. Both applications again result in reduced field resources per population and the ability to evaluate larger numbers of populations within a given field unit.

[0136] Double haploid (DH) plants provide plant breeders with an invaluable tool, especially for generating inbred lines. Because homozygous lines are generated virtually instantly, significant time is saved, eliminating the need for multiple generations of conventional inbreeding.

[0137] In particular, because DH plants are completely homozygous, they are well-suited for quantitative genetics studies. Additive variance and additive x additive genetic variance can be estimated from DH populations. Other applications include the identification of ectopic dominance and linkage effects. For breeders, DH populations have been particularly useful in QTL mapping, cytoplasmic transformation, and trait introgression. Furthermore, they are valuable for plant breeding programs in testing and evaluating homozygous lines. All genetic variation is inherited from the offspring of breeding hybrids, which enhances selection gains.

[0138] However, it is well known in the art that DH generation processes are inefficient and can be very labor-intensive. While double haploid plants can occur spontaneously in nature, this is extremely rare. Most research and breeding applications rely on artificial methods of DH generation. The initial step involves haploidizing the plant, which results in a population containing haploid seeds. A non-homozygous line is then crossed with an inducer parent to produce haploid seeds. Seeds with haploid embryos but normal triploid endosperm advance to the second stage. That is, haploid seeds and plants are any plants with haploid embryos, regardless of the ploidy level of the endosperm.

[0139] After selecting haploid seeds from the population, the selected seeds undergo chromosome doubling to produce double haploid seeds. Spontaneous chromosome doubling in a cell lineage will result in the production of normal gametes or unreduced gametes from a haploid cell lineage. The rate of diploidization can be increased by applying compounds such as colchicine. Colchicine binds to tubulin and prevents it from polymerizing into microtubules, thereby inhibiting mitosis in metaphase, and can be used to increase the rate of diploidization (i.e., chromosome doubling). These chimeric plants self-pollinate to produce diploid (double haploid) seeds. These DH seeds are cultured and subsequently evaluated and used for hybridization testcross production.

[0140] However, even with the development of methods to increase the frequency of DH production, including colchicine treatment, methods for producing DH seeds generally remain low in efficiency. Prominent problems include low haploid seed yields, reduced gamete viability, resulting in decreased self-pollination of DH plants and insufficient DH seed yields for breeding applications.

[0141] The seed sampling systems (e.g., System 10, etc.) and related methods (including the described analytical methods and seed breeding methods) of this disclosure represent an advancement in breeding applications by facilitating selection potential at both the haploid and diploid seed stages. For example, the seed sampling systems (e.g., System 10, etc.) and related methods (including the described analytical methods and seed breeding methods) of this disclosure can provide high-throughput sampling of entire populations of haploid seeds and allow for subsequent analysis of samples removed from the seeds. This can also provide high-throughput stacking of entire populations of double haploid seeds. The presence or absence of one or more traits in the sample indicating at least one genetic or chemical trait can be analyzed, and based on the results of said analysis, one or more individual double haploid seeds can then be selected, and plants or plant tissues can be cultured from the selected double haploid seeds.

[0142] The seed sampling system (e.g., System 10, etc.) and related methods (including the described analytical methods and seed breeding methods) disclosed herein may also include operations associated thereto for analyzing one or more characteristics of the seed, such as genetic markers, transgenes, markers linked to or diagnosing transgenes, characteristics related to event performance, event evaluation, and trait integration, to determine whether the seed is in a haploid or diploid state and / or to select preferred genotype and phenotypic categories for doubling.

[0143] In another embodiment, the seed sampling system (e.g., System 10, etc.) and related methods (including the described analytical methods and seed breeding methods) of this disclosure can be used with operations for determining linkage phases. By using seed endosperm tissue derived from diploid plants, a genotyping system capable of detecting different allele frequencies in a DNA sample can be used to determine the parental marker haplotype. Since the endosperm tissue is triploid with two copies derived from the female gamete, the linkage phase of the parental line can be obtained by dissecting the genotypes of heterozygous offspring. DNA samples from the endosperm tissue allow for the determination of the ploidy level of genetic markers. The diploid ploidy level in genetic markers indicates maternal inheritance, and the haploid ploidy level in genetic markers indicates paternal inheritance.

[0144] Furthermore, differential allele frequency data can be used to infer genetic linkage maps, but unlike methods that require haploid material, the aforementioned allele frequency calls are used. Determining genetic linkage maps has immense utility in the context of haplotype characterization and mapping of marker (or haplotype)-trait associations. This is particularly robust based on single-seed-pair stacked seeds and is therefore well-suited for use in conjunction with the seed sampling systems of this disclosure (e.g., System 10, etc.) and related methods (including the described analytical methods and seed breeding methods).

[0145] In another embodiment, the seed sampling system of this disclosure (e.g., System 10, etc.) and related methods (including the described analytical methods and seed breeding methods) can be further used in conjunction with an assay for predicting embryo conjugativity of a specific target gene (GOI). This assay predicts embryo conjugativity based on the ratio of the relative copy number of the GOI to the internal control (IC) gene per cell or per genome. Generally, this assay uses IC genes with known conjugativity, such as homozygous at the locus (two IC copies per diploid cell), to standardize the measurement of GOI. The ratio of the relative copy number of IC to GOI predicts the GOI copy number in the cell. In homozygous cells, for any given gene (or unique genetic sequence), the gene copy number equals the cell's ploidy level because the sequence is present at the same locus in all homologous chromosomes. When a cell is heterozygous for a particular gene (or hemizygous in the case of a transgenic gene), the gene copy number will be lower than the cell's ploidy level. If no GOI is detected, the cell is empty at the locus, as may be the case for negative segregators of a transgenic event or in a mutagenic population. Therefore, the conjugation of a cell at any locus can be determined by the number of gene copies in the cell.

[0146] In one particular embodiment, the seed sampling system (e.g., System 10, etc.) and related methods (including the described analytical methods and seed breeding methods) of this disclosure can be used in conjunction with assays for predicting conjugation in maize embryos. In maize seeds, the endosperm tissue is triploid, while the embryo tissue is diploid. Endosperm copy number reflects embryo conjugation: homozygous (positive or negative) endosperm is associated with a homozygous embryo, and heterozygous endosperm (GOI copy number 1 or 2) reflects a heterozygous (GOI copy number 1) embryo. For IC, a homozygous endosperm will contain three IC copies. The endosperm GOI copy number can range from 0 (homozygous negative embryo) to 3 (homozygous positive embryo); and an endosperm GOI copy number of 1 or 2 is found in seeds in which the embryo is heterozygous for GOI (or hemizygous for GOI if GOI is transgenic). The endosperm GOI copy number (which can range from 0 to 3 copies) can be determined by the ratio of the endosperm IC copy number to the endosperm GOI copy number (the ratio can range from 0 / 3 to 3 / 3, i.e., from 0 to 1), which can then be used to predict embryo conjugation.

[0147] As is known in the art, the copy number of GOI or IC can be determined by any convenient measurement technique used to quantify copy number. Examples of suitable measurements include, but are not limited to, real-time... PCR (Applied Biosystems, Foster City, CA) and (Third Wave Technologies, Madison, WI) assay. Preferably, such assays are developed in a manner that makes the amplification efficiencies of both the IC and GOI sequences equal or very similar. For example, in real-time In PCR assays, signals from single-copy GOI (determining heterozygous source cells for GOI) will be detected one amplification cycle later than signals from double-copy IC, because the amount of GOI is half that of IC. For the same heterozygous sample, The determination will measure a GOI / IC ratio of approximately 1:2 or 0.5. For samples where both GOI and IC are homozygous, the GOI signal will be detected simultaneously with the IC signal. Furthermore, the Invader assay will measure a GOI / IC ratio of approximately 2:2 or 1.

[0148] These guidelines apply to any polyploid or haploid cell (such as pollen cells) because the copy number of GOI or IC remains proportional to the cell's genome copy number (or ploidy level). Therefore, these conjugation assays can be performed on triploid tissues, such as maize endosperm. Furthermore, the copy number of GOI can be measured to be more than 2 copies, or values ​​that are numerically different from the cell's ploidy. The methods remain applicable to detecting GOI in polyploids in some transgenic events with >2 copies of the inserted transgene, after GOI replication via translocation, when GOI is present on autonomously replicating chromosomes or plasmids, and in other cases.

[0149] In plant breeding, determining conjugation at one or more loci is useful for evaluating inbreeding levels (i.e., the degree of gene fixation), segregational aberrations (i.e., in transgenic germplasm, maternal genetic testing, or for loci affecting gamete fitness), and dissociation levels (i.e., the relative proportions of homozygotes and heterozygotes). Similarly, the degree of conjugation at one or more loci can be used to estimate heterosis and whether a particular seed batch meets commercial or regulatory standards for sale as certified hybrid seeds. Furthermore, in transgenic germplasm, knowing ploidy or copy number is useful for distinguishing quality events and aiding in trait integration strategies.

[0150] In another embodiment, the seed sampling system (e.g., System 10, etc.) and related methods (including the described analytical methods and seed breeding methods) of this disclosure can be used in conjunction with operations for improving the ability to monitor frequency changes of one or more genetic traits in one or more germplasm banks, said genetic traits including markers, alleles, and haplotypes. Methods known in the art for comparing the frequencies of genetic markers between recently acquired populations and their ancestral lines to identify those genetic loci whose frequencies increase over time are known (US Patent Nos. 5,437,697 and 5,746,023). Those loci whose inferred frequencies exceed expected allele frequencies have been selected. Furthermore, given that yield is a primary selection criterion in breeding procedures, those alleles that are expected to become increasingly frequent are likely associated with yield.

[0151] In one particular implementation, the seed sampling system (e.g., System 10, etc.) and related methods (including the described analytical methods and seed breeding methods) of this disclosure can be used in conjunction with operations for haplotype-assisted breeding. It is possible to identify haplotypes that deviate from expected haplotype frequencies by comparing haplotype frequencies in emerging superior lines with haplotype frequencies in ancestral superior lines (as determined via pedigree analysis). Furthermore, by evaluating haplotype effect estimates, it is also possible to link increased frequency haplotypes to phenotypic outcomes for a set of agronomic traits. The haplotype composition of individual seeds sampled from multiple seeds can be determined using genetic markers, and seeds with preferred haplotypes can be selected and promoted. Therefore, this technique enables more informed breeding decisions and the establishment of superior line development programs.

[0152] As described above, the seed sampling system 10 (and its various components) can be controlled (and / or coordinated) by a central control system (widely, a computing device). In connection with this, Figure 22 An exemplary relationship between a seed sampling system 10 and a corresponding control system 200 is illustrated. As shown, the seed sampling system 10 is connected to (and communicates with) the control system 200 via a network 202 to facilitate the aforementioned communication and interaction. Furthermore, in this context, the network 202 may include, but is not limited to, a local area network (LAN), a wide area network (WAN) (e.g., the Internet), a mobile network, a virtual network, and / or other suitable public and / or private networks capable of supporting communication between the seed sampling system 10 and the control system 200, or any combination thereof. Alternatively, as... Figure 22 As shown by the dashed line, the seed sampling system 10 can be directly coupled to the control system 200 (and communicate with the control system) via, for example, a wired connection (for example, the control system 200 can be a component of the seed sampling system 10).

[0153] Figure 23An exemplary computing device 300 is shown that can be used in conjunction with a seed sampling system 10 and a control system 200. The computing device 300 may include, for example, one or more servers, workstations, personal computers, laptops, tablets, smartphones, etc. Furthermore, the computing device 300 may include a single computing device, or it may include multiple computing devices that are closely adjacent to or distributed within a geographical area, provided that the computing devices are specifically configured to operate as described herein. Figure 22 In an exemplary embodiment, each of the seed sampler system 10 and the control system 200 may be considered to be included and / or implemented in at least one computing device consistent with computing device 300. However, as described below, this disclosure should not be considered limited to computing device 300, as different computing devices and / or arrangements of computing devices and / or arrangements of components associated with such computing devices may be used.

[0154] refer to Figure 23 An exemplary computing device 300 includes a processor 302 and a memory 304 connected to (and in communication with) the processor 302. The processor 302 may include one or more processing units (e.g., in a multi-core configuration). For example, the processor 302 may include, but is not limited to, a central processing unit (CPU), a microcontroller, a reduced instruction set computer (RISC) processor, an application-specific integrated circuit (ASIC), a programmable logic device (PLD), a gate array, and / or any other circuitry or processor capable of implementing the functions described herein.

[0155] As described herein, memory 304 is one or more means that allow data, instructions, etc., to be stored therein and retrieved therefrom. Memory 304 may include one or more computer-readable storage media, such as, but not limited to, dynamic random access memory (DRAM), static random access memory (SRAM), read-only memory (ROM), erasable programmable read-only memory (EPROM), solid-state devices, flash drives, CD-ROMs, thumb drives, floppy disks, magnetic tapes, hard disks, and / or any other type of volatile or non-volatile physical or tangible computer-readable media. Memory 304 may be configured to store, but not limited to, various types of data (and / or corresponding data structures) described herein. Furthermore, in various embodiments, computer-executable instructions may be stored in memory 304 for execution by processor 302 to cause processor 302 to perform one or more functions described herein, such that memory 304 is a physical, tangible, and non-transitory computer-readable storage medium. Such instructions generally improve the efficiency and / or performance of processor 302 and / or other computer system components configured to perform one or more of the various operations described herein. It should be understood that memory 304 may include a variety of different memories, each of which is implemented in one or more functions or processes described herein.

[0156] In an exemplary embodiment, the computing device 300 further includes a presentation unit 306 coupled to (and in communication with) the processor 302 (however, it should be understood that the computing device 300 may include output devices other than the presentation unit 306). The presentation unit 306 outputs information to the user of the computing device 300 as needed. Furthermore, various interfaces (e.g., those defined by web-based applications) may be displayed on the computing device 300, and particularly on the presentation unit 306, to display such information. The presentation unit 306 may include, but is not limited to, a liquid crystal display (LCD), a light-emitting diode (LED) display, an organic LED (OLED) display, an "electronic ink" display, a speaker, etc. In some embodiments, the presentation unit 306 may include multiple devices.

[0157] Furthermore, the computing device 300 includes an input device 308 that receives input from a user of the computing device 300. The input device 308 may include a single input device or multiple input devices. The input device 308 is coupled to (and communicates with) a processor 302 and may include one or more of, for example, a keyboard, a pointing device, a mouse, a touch-sensitive panel (e.g., a touchpad or touchscreen), another computing device, and / or an audio input device. Additionally, in various exemplary embodiments, a touchscreen, such as a touchscreen included in a tablet computer, smart device, or similar device, may serve as both a presentation unit and an input device.

[0158] Furthermore, the computing device 300 shown also includes a network interface 310 coupled to (and in communication with) the processor 302 and the memory 304. The network interface 310 may include, but is not limited to, a wired network adapter, a wireless network adapter, a mobile network adapter, or other means capable of communicating with one or more different networks (including network 202) and / or the seed sampler system 10. Additionally, in some exemplary embodiments, the computing device 300 may include a processor 302 and one or more network interfaces incorporated into or incorporated with the processor 302.

[0159] The foregoing description of embodiments has been provided for illustrative and descriptive purposes. It is not intended to be exhaustive or limiting of the invention. Individual elements or features of a particular embodiment are generally not limited to the particular embodiment described, but are interchangeable where applicable and can be used in selected embodiments, even if not specifically shown or described. This can also be varied in many ways. Such variations should not be considered as departing from the invention, and all such modifications are intended to be included within the scope of the invention.

[0160] Exemplary embodiments have been provided to make this disclosure as extensive and fully communicative of the scope to those skilled in the art. Numerous specific details, such as examples of specific parts, components, and methods, are set forth to provide a thorough understanding of embodiments of this disclosure. It will be apparent to those skilled in the art that specific details are not required, that exemplary embodiments may be embodied in many different forms, and that exemplary embodiments should not be construed as limiting the scope of this disclosure. In some exemplary embodiments, well-known processes, well-known apparatus structures, and well-known technologies are not described in detail.

[0161] The specific dimensions, materials, and / or shapes disclosed herein are exemplary in nature and do not limit the scope of this disclosure. The disclosure of specific values ​​and ranges of values ​​for a given parameter herein does not exclude other values ​​and ranges of values ​​that may be useful in one or more instances disclosed herein. Furthermore, it is contemplated that any two specific values ​​of the aggregate parameter described herein can define endpoints of a range of values ​​that may be applicable to the given parameter (i.e., the disclosure of a first and second value of a given parameter can be interpreted as disclosing that any value between the first and second values ​​may also be applicable to the given parameter). For example, if parameter X is exemplified herein as having a value A and also exemplified as having a value Z, it is contemplated that parameter X may have a range of values ​​from about A to about Z. Similarly, the disclosure of two or more ranges of values ​​for a parameter (whether such ranges are nested, overlapping, or distinct) includes all possible combinations of the range of values ​​that may be required using the endpoints of the disclosed ranges. For example, if parameter X is exemplified in this document as having a value in the range of 1-10, 2-9, or 3-8, it is also conceivable that parameter X may have other ranges of values, including 1-9, 1-8, 1-3, 1-2, 2-10, 2-8, 2-3, 3-10, and 3-9.

[0162] The terminology used herein is for the purpose of describing particular exemplary embodiments only and is not intended to be limiting. As used herein, the singular forms “a / an” and “described” may also include the plural forms unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” and “having” are inclusive and therefore specify the presence of said features, integers, steps, operations, elements, components, and / or groups thereof, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Unless specifically specified as performed in order, the method steps, processes, and operations described herein should not be construed as necessarily requiring performance in the specific order discussed or shown. It should also be understood that additional or alternative steps may be employed.

[0163] When an element or layer is referred to as “on another element or layer,” “joined to,” “connected to,” or “coupled to” another element or layer, it may be directly on, joined to, connected to, or linked to another element or layer, or there may be intermediate elements or layers present. In contrast, when an element is referred to as “directly on another element or layer,” “directly joined to,” “directly connected to,” or “directly coupled to” another element or layer, there may be no intermediate elements or layers present. Other terms used to describe relationships between elements should be interpreted in a similar manner (e.g., “between” vs. “directly between,” “adjacent” vs. “directly adjacent,” etc.). As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0164] While the terms first, second, third, etc., may be used herein to describe different elements, components, seeds, members, and / or segments, these elements, components, seeds, members, and / or segments should not be limited by these terms. These terms are used only to distinguish one element, component, seed, member, or segment from another. Unless the context clearly indicates otherwise, terms such as “first,” “second,” and other numerical terms used herein do not imply order or sequence. Therefore, without departing from the teachings of the exemplary embodiments, the first element, component, seed, member, or segment discussed below may be referred to as the second element, component, seed, member, or segment.

[0165] For ease of description, spatially relative terms such as “inside,” “outside,” “below,” “below,” “lower,” “above,” “upper,” etc., are used herein to describe the relationship between one element or feature and another element or feature as shown in the figures. Spatially relative terms are intended to cover different orientations of the device in use or operation other than those depicted in the figures. For example, if the device in the figure is flipped, an element described as “below” or “below” other elements or features will be oriented as “above” other elements or features. Thus, the exemplary term “below” can cover both above and below orientations. The device may be oriented in other ways (rotated 90 degrees or other orientations), and therefore the spatially relative descriptive terms used herein are explained.

Claims

1. An automated method for removing tissue samples from seeds, the method comprising: The first seed is held in the first pair of arms of the seed clamping assembly of the sampling module of the automated seed sampling assembly, and the second seed is held in the second pair of arms of the seed clamping assembly of the sampling module. The first seed is moved toward the sampler of the sampling module associated with the first sampling position of the sampling module by means of the seed clamping assembly; Remove the tissue sample from the first seed at the first sampling location; While removing the tissue sample from the first seed at the first sampling position of the module, an actuated blow-out nozzle guides air through the second sampling position toward a collection port located near the sampler at the second sampling position; and residual seed tissue is drawn into the collection port by a negative pressure airflow at the collection port to remove the residual seed tissue from the second sampling position of the sampling module. and The second seed is then moved toward the sampler of the sampling module associated with the second sampling position of the sampling module by the seed clamping assembly, and the tissue sample is removed from the second seed at the second sampling position, while residual seed tissue is removed from the first sampling position using the blow-out nozzle and collection port provided at the first sampling position.

2. The automated method of claim 1, further comprising receiving the tissue sample removed from the first and second seeds in a sample plate and receiving the first and second seeds from which the tissue sample has been removed in a seed tray.

3. The automated method of claim 2, further comprising assigning an identifier to each of the first and second seeds and the tissue sample removed from the seeds, whereby the identifier is available for subsequent identification of the seeds in the seed tray and the corresponding tissue sample in the sample plate.

4. The automation method as described in claim 1, further comprising: The tissue sample removed from the first seed at the first sampling position in the sampling module is delivered to the sample collection plate via the nozzle block; and The residual seed tissue removed from the second sampling position of the sampling module is transported by the nozzle block to the purge block for processing.

5. The automated method of claim 1, further comprising determining the position of the first seed in the sampling module by means of at least one sensor disposed near the first sampling position.

6. The automation method of claim 1, further comprising: Pick the first seed from a plurality of seeds; Engage the first seed picked by the single sorting with the holding member of the automated seed delivery assembly; and The first seed is oriented at the holding member and moved to a first position of the sampling module, thereby facilitating the removal of tissue sample from the seed at the first sampling position.

7. The automated method of claim 1, further comprising analyzing one or more characteristics of the tissue sample, and selecting or not selecting the seeds from which the tissue sample has been removed based on the presence of the one or more characteristics in the tissue sample.

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