AU2014200607B2 - Systems and methods for processing hybrid seed - Google Patents

Abstract

A. method is provided for testing individual seeds in a population for a trait of interest. In various embodiments, the method comprises providing a population of seeds, separating, via an automated seed singulator, individual seeds from the population of seeds, and removing a tissue sample comprising cells with nucleic acids from the individual separated seeds, via an automated sampling assembly, while preserving the germination viability of the seed, The method additionally includes analyzing nucleic acids extracted from each tissue sample to determine the presence of at least one genetic marker indicating the presence of a genetic trait of interest; and selecting particular seeds front the individual sampled seeds based upoi the determination of the presence of the at least one genetic marker in the particular seeds.

Description

2014200607 05 Feb 2014 ~1 A- CROSSTFEFERBNCE TO RELATED applications

This application is a ‘divisional’ application derived ftom Australian Patent Application No. 2008296436, which is the national phase of International Application No. ΡΟΤ/ϋ32668/0746()8, elapaing pdofity of U,B7^ 60/968,800. filed on August 29, 2009, the entire text of which are hereby incofoorated herein by reference.

FIELD

The present disclosure relates to the field of pi ant breeding, Mtnre sped fically, this disclosnie provides systems and methods for producing various hybrid seed.

BAGR.GROTIHD

The- statements in this section ntetely ptoyidc background information related to the present disclosure and may not constitute prior art.

Various hybrid seed is currently produced byremoving male flower parts of the female parent by hand~emaseu|ation^ and: then harKl^olbnaiing the female flowers with pollen from a male parent. This mdhod iSilabor intensive and expensive, and limits seed production to areas where the Use of hand labor is available and cost-effective. For example, gynecious plant types have been developed for use as female pare nts in some types of melons, and i f strongly expressed, these; types do not require hand manipulations. However, the gynecious trait is complexly inherited and incompletely penetrant, making it difficult to breed and use.

Genetic malfosteiilliy controlled hi single recessive nuclear genes has been identified in cucurbits and: other fruit and vegetable bearing plants. This trait cotdcl be used to develop male-sterile female parents that would hot require Rower removal or hand emasculation, and ii would allow the use of bees tor 2014200607 05 Feb 2014 ~2- poliination. However, male-sterile Sines always segregate lor sterile and fertile giants because they must be maintained by pollinating male-sterile plants fms/ms) wit) heterosygous» isogenic male-fbrtiie plants (Ms/ms). Thus, the use of nuclear genic male-sterility requires that the male-fertile segregants be 5 removed from the female parent rows in hybrid seed production fields. This process: is labor intensive and often ineffective because the male-fertile plants are difficult to Identify in the field, For this reason, previous efforts by Seed companies to develop this trait were abaridoned.

The present disclosure addresses heeds in the art for improved methods .10 of producing hybrid seeds using high-throughput, nondestructive seed sampling systems, mwMmf

The present disclosure relates to systems and methods for facilitating germplasm imptayement activities In seeds through the use of high-throughput, 15 nondestructive seed sampling, With automated, nondestructive sampling, it is possible to test individual seeds in a population, and select only the seeds that possess one or more desired characteristics. This allows for new and more efficient methods for germplasm improvement and management, which lead to improved breeding populations, 20 in various embodiments, the present disclosure provides for an automated

System for lie high-throughput sampling of seeds. The system comprises a seed loading station fpr separating individual seeds from a plurality of like seeds in a bulk seed bin; an orientation subsystem for receiving the individual seeds ten the seed loading station and orienting the individual seeds; a sampling 25 subsystem for removing a tissue sample from the individual seeds* a seed and sample transport subsystem 1pr conveying the seed between the: sampiirig subsystem arid a selected: well in a seed collection tray after the seed has had a 2014200607 05 Feb 2014 :.-3- tissue sampie removed, and for conveying the tissue sample tom the sampling subsystem to a selected weii.in a sample collection tray. in other embodiments, the present disclosure provides lor an automated, higb-througbput method for extracting sample material for testing tom a 5 population of seeds. The method comprises separating individual seeds from the population of seeds and orienting the separated seeds in a desired orientation Such that a Tip1 of each seed, containing the embryo of each respective seed, is placed in a particular orientation while preserving the germinatiph viability of the seed. The oriented seeds are then conveyed to a sampling retention fixture and 10 a sample of seed tissue is removed fcm each seed, while preserving the germination viability of each seed. Each seed IS then conveyed to a selected well in a seed collection tray after the seed has been sampled and the seed tissue sampie is ODoveyed to a selected well in a sampie collection tray. in sttit other embodiments, the present disclosure provides for an 15 automated method for the high-throughput sampling of seeds, the method includes separating individual seeds from a plurality of ilke seeds at a seed loading station of an automated seed processing system. The method additionally includes receiving and orienting the individuai seeds, while preserving the germination viability of each seed, at an orientation subsystem of 20 the automated seed processing system. The method further includes removing a tfesue sarnpie fiem the Individual seeds, while preserving the germination viability of each seed, at a sampling subsystem of the automated seed processing system. Still further, the method includes depositing each seed into a selected well in a seieeied one of a plurality of seed collection trays after toe seed has had 25 a tissue sampie removed and depositing each tissue sample into a selected well in a selected one of a plurality of sampie collection trays, utilizing a seed and sampie transport subsystem of if# automated seed processing system. The Ο <Ν 2014200607 05 Feb -4-,. method further yet includes storing in a database the selected well in the selected sempfert sample was deposited and the seiected well in the Selected seed collection tray into which each corresponding sampled seed was deposited, such that each sample and corresponding seed 5 from which the sample was removed can be tracked to pre-select seeds of Interest. in yet other ernbodiments, fie present disclosure provides lor a method for infrogressing male sterility into a seed. The method comprises providing a population of seeds, removing a tissue sample composing ceils with nucleic 10 acids from each seed in the population, analyzing the nudesc acids exacted from each seed for the presence Of at least ope genetic marker indicating foe presence of a male-sterile gene, selecting seeds from the population based upon foe presence of foe male-sterile marker* and cultivating a fertile plant from foe seed. 15 In still yet other embodiments, the disclosure provides for a female parent of a fruit or vegetable hybrid wherein foe female parent contains at ieast one nuclear mate sterile gene. in the various embodiments of the present disciosurejhe samples may be analyzed for one or more characteristics indicative of at least one genetic trait. M Examples of such characteristics may include a genetic marker, a single nucleotide polymorphism, a simple sequence repeat, a restriction fragment length polymorphism, a haplotype, a tag SlsiP, an alleles Of a genetic marker, a gene, a DMA-derived sequence, an RNA-denved sequence, a promoter, a 5’ untranslated region of a gene, a 3' untranslated region of a gene, microRNA, 25 siRNA, a dTL, a satellite marker, a transgene, mRNA, ds mRNA, a transcriptional profifej and a niethylation pattern.

Further areas of applicability of the present teachings will become a pparent frorn the description provided herein. It should be understood that Hie description and specific examples are intended lor purposes of illustration only and are not intended to llrtiit thestp^ 2014200607 05 Feb 2014

5 BRIEF DESCRIPTION GF THE DRAWINGS

The drawings described herein are forillustrationpunposes only and are not intended to limit the scope of the present disclosure in any wayi

Figure 1 is a block diagram of an automated seed processing system for nondestructiyely sampling seeds, in accordance with various embodiments of the to present disclosure.

Figure 2 is an isometric view of the seed processing system shown in Figure 1, in accordance with various embodiments of the present disclosure.

Figure M Is 1foht yiew of an orientation pedestal of the seed processing system shown in Figufie 1, in accordance with various embodiments of the 15 present disclosure.

Figure 2B Is front view of an orientation pedestal of the seed processing system shown in Figure 1, in accordance with various other embodiments of the present disclosure.

Figure 2C is front view of an orientation pedestal erf the seed processing 20 system shown in Figure 1, in accordance with still other various embodiments of the present disclosure.

Figure 2D is front view of an orientation pedestal of the seed processing system shown in Figure 1, in accordance with yet other various embodiments of the present disclosure. ~β~ 2014200607 05 Feb 2014

Figure 2E is front view of an orientation pedestal of the seed processing system shown in Figure 1, in accordance with various still yet other embodiments of the present disclosure.

Figure 3 is a side view of the seed processing system shown in Figures 1 5 and Z, in accordance with various embod iments of the present invention .

Figure 4 is an isometric view of a seed loading station, a seed orientation subsystem and a seed sampling subsystem of the seed: processing system shown in Figures tf 2 and 3, in accordance with various embodiments of the present disclosure. 10 Figure 4A is a side view of the seed loading station of the seed sampling subsystem shown in Figures 1,2 and 3, iiiusfrating a seed singulator and diverter assembly, in accordance with various embodiments of the present disciosure.

Figure 5 is an Isometric view of the seed sampling subsystem shown in Figures 1, 2, 3 and 4, in accordance with various embodiments of the present 15 disclosure.

Figure 6 is a front view of a linear actuated seed sampling assembly of the seed sampling subsystem shown in Figure 1, in accordance with various embodiments of the present disciosure.

Figure 7 is a front view of the linear actuated seed sampling assembly 20 shown in Figure 6, in accordance with various other embodiments of the present disclosure.

Figure 6A Is a front View of a sampling retention fixture of the seed sampling subsystem shown in Figure 1, in accordance with various embodiments of the present disclosure^ 7 2014200607 05 Feb 2014

Figure 8B is a front view of a sampling retention fixture of the seed sampling subsystem shown in Figure 1, in accordance with various other embodiments of the present disclosure.

Figure 8C is a front view of a sampling retention fixture of the seed sampling subsystem shown in Figure 1, in accordance with still other various embodiments of the present disclosure^

Figure 8D is a front view of a sampling retention fixture of the seed sampling subsystem shown in Figure I, maccordanee with yet other various embodiments of the present disclosure.

Figure 8E is an isometric view of a sampling retention fixture of the seed sampling subsystem shown in Figures 1, 2, 3 and 4, in accordance with various embodiments of the present disclosure.

Figure 8F is a cross-sectional view of the sampling retention fixture shown in Figure :8¾ along line A-A, in accordance with various embodiments of the present disclosure.

Figure 9 is a block; diagram of a master control system for the seed processing system shown in Figure 1, in accordance with various embodiments of the present disclosure.

Figure 10 is ail image of agarose gel showing: resolution of alleles at the marlmr linked to the ms3 gene.

Figure il illustrates genetic linkage groups containing the ms3 and ms4 genes showing positions of the ms genes relative to linked molecular markers.

Figure 12 is an agarose gel image showing FCR products from reaction mixes using BN A isolated from melon seed tissue samples. i<ACVVV>- -8- 2014200607 05 Feb 2014 m$mm description

Tie following: description is merely exemptary in nature and is not intended to limit thepresent disclosure, application, or usds.

The present;disclosure provides tor novel methods to facilitate germ plasm 5 improvement activities in seeds, such as broccoli, cabbage, carrot, cauiifidwer, Chinese cabbage; cucumber, dry bean,.eggplant,, fennel, garden beans, gourd, Jeek, lettuce, melon, okra, onion, pea, pepper, pumpkin, radish.. spinach, squash, sweet com, tomato and watermelon, using high-throughput, hohdestmctive: sampiing of seeds· The methods are useful in analyzing Seeds in order to 10 identify and detect seeds comprising one or more desired traits, markers, and genotypes, In One aspect of the disclosure, the analytical methods allow individual seeds that are present in a batch or a bulk population of seeds to be analyzed such that the chemical and/or genetic characteristics of the individual seeds can be determined 15 Apparatus, systems and methods tar the high-throughput, nondestructive sampling of seeds have been described. For example, commonly owned U.S. Patent Application Serial No. 11/213,430 (rlled August 26, 2003); U.S, Patent Application Serial No. 11/213,431 (filed August 26, 2005);: U.S. Patent

Application Serial No. 11/213,432 (filed August 26, 2005); U.S, Patent 20 Application Serial No, 11/213,434 (filed August 26, 2005); U.S. Patent

Application Serial NO. 11/213,435 (filed August 28, 2005); U.S. Patent

Application Serial No. 11/680,180 (filed February 27, 2007); and U.S. Patent Application Serial No. 11/680,611 (fifed February 27, 2007), which are incorporated herein by reference in their entirety, disciose apparatus and 25 systems for the automated sampiing of seeds; as well as methods of sampling, testing and bulking seeds. interring to Figure 1, tee present disclosure provides an automated seed processing system 10 structured and operable to facilitate germpiasm improvement activities in selected seeds prough the use of high-throughput, nondestructive seed sampling. More particularly, as described beiow, the seed 5 processing system 10 is stasctured and; operable to test individual seeds in a population, and select only tee seeds that possess one or more desired characteristics, thereby providing efficient methods for germpiasm improvement and management leading to improved breeding populations. 2014200607 05 Feb 2014

In various embodiments, the seed processing system 10 generally 10 includes a seed loading station 100 that is structuredand operable to separate individual seeds from a plurality of like seeds, and an orientation subsystem 200 that receives the individual seeds pirn tee seed loading station 100 and orients each seed in a desired orientation for sampling, as described below, Addifwnaiiy, the seed processing system; 10 generally includes a sampling 15 subsystem 300 Pat is structured and operable to remove a sample, e.g., a tissue sample, from each seed, and a seed and sample transport subsystem 400 that is structured and operable for Conveying each seed and the respective sample from the sampling station 300 to a seed collection assembly; 500 and a sample collection assembly 600, respectively, fyipte particularly, the seed and Sample 20 transport subsystem 400 is structured and operable to convey each sampled seed to; one of a plurality of seed collectidn trays 504 (exempianiy shown in Figure 2), and convey each respective sample to one of a plurality of sample collection trays 604, e.g., micropiates (exemplarily shown in Figure 2).

The seed processing system 10 additidnaily includes a master control 25 system 700 that is structured and operable to coniro! all the operations of the seed processing system 10, That is, the master control system 700 simultaneously controls and coordinates the operations of the seed loading -10- 2014200607 05 Feb 2014 station 100» the orientation subsystem 200, the sampling subsystem 300, the seed and sample transport subsystem 400, and the seed and sample collection assemblies 500 and 600 to carry out the separating of the seeds tern the plurality of seeds, the orienting of each seed; in a desired orientation, the 5 removing of a sample from each seed, and the conveying each seed and respective sample of the cormsponding seed and sample trays 504 and; 604, as described; below, it should be understood that the various embodiments of die seed processing system 10, exempiariiy illustrated and described herein, include 10 various braces, beams, platforms, pedestals, stands, etc,, to which various components, devices, mechanisms, systems* subsystems, assemblies and subassemblies described herein are coupled, wnnected and/or mounted. Although such braces; beams, platforms, pedestals, stands, etc», are necessary to the construction of various embgdimerits erf the seed processing system 10, 15 description Of their placement, orientation and interconnections are not heCdsdafy for one skilled in the art to easily and fuliy comprehend the structure, function and operation Of the various embodiments of the seed processing system 10. Moreover, such braces, beams, platforms, pedestals, stands, etc., areclearly illustrated in various figures and, as such, their placement, orientation 20 and fn^roonheefions are easily understood by one skilled in the art. Therefore, for simplicity; such braces, beams, platforms, pedestals, stands, etc., will be referred to herein merely as system support stru^dris, abseirf forther^description of their placement, orientation and interconnections. Additionally, certain figures described and illustrated herein may have portions of the system support 25 structures removed, i.e., not shown; in order to more clearly illustrate the various embodiments of the seed processing system 10.

Referring now to Figures 2 and 3, in various embodiments, the loading -Π- 2014200607 05 Feb 2014 station 100 can comprise a seed singu later 104, and the orientation subsystem 200 dan comprise it plurality of rotatable orieniation pedestals 204 and one or more imaging devices 208 mounted to system support structure above tie orientation pedestals 204, Additionally* the sampling subsystem 300 can 5 comprise a plurality of corresponding sample retention: fixtures 304, and: the seed and sample transport subsystem 400 can comprise a plurality of diverter assemblies 404 mounted beneath the sampling retention fixtures 304,

Referring now to Figures 2, 3 and 4, in various embodiments, the seed singutator· 104 comprises a seed bin 110, a separating wheel 112 and a seed Id singulation chamber 118 located between a bottom (not shown) of theseed bin 110 and the separating wheel 112. The seedsihgulaior 104 additionally includes a motor 114, e g., and indexing motor, coupled to the separation wheel 112 to eontoliably rotate the separating wheel 112, as controlled by the master control system 700, Moreover, the separating wheel 112 is mounted forroiafion in a 15 vertical piarie Such that, during operation, a face (not shown) ofthe separating wheel 112 rotates adjacent an open side Of the seed singulation chamber 116 and also adjacent a seed collector 120 located between the seed bin 110 and the separating wheel 112. The face of the seed separating wheel 112 includes a plurality of recessed ports (not shown) that are circumferentially spaced apart 20 around a perimeter portion of the separating wheel 112. Each recessed port extends thfbugh the face of the separating wheel 112 and is communicatively coupled to a vacuum system (not shown) such that a vacuum can be provided at each of the recessed ports.

In various embodiments* to initiate operation of the: seed processing 25 system 10, a plurality of seeds to be sampled and tested are placed in an interior reservoir of the seed bin 110. Via gravity, forced air and/or a vacuum, the seeds deposited into the seed bin interior chamber are pphtrollably funneled into the singulation chamber 118 and contact the face Of the Separating: wheel 112. .--12- 2014200607 05 Feb 2014

Additionaily, a vacuum is provided to at least some of the recessed ports, e.g., a vacuum is provided to at feast the recessed ports traveling adjacent the open side of the seed singulation chamber 116 as the separating wheel 112 rotates.

To separate seeds, substantially one-at-a-time, from the plurality of seeds 5 deposited into the seed bin 110^ i.e., singufate the seeds, the separating wheel 112 is ineremeotaliy rotated, via ; 114. As the separating wheel 112 Is mtated each recessed portsequentialfy travels past the open side of the seed singulation chamber 116, extracts at least one seed, and deposits eadh seed into the seed effector 120. That is, as the separating wheel 112 incrementally 10 rotates and the recessed ports incrementally pass adjacent the singulation chamber 118, individual ones of the seeds in contact with theseparating wheel face are picked up and held at each recessed port by the vacuum provided at tie respective recessed ports. As tie· separating Wheel 112 continues to incrementally rotate, tie seeds are carried out of the singulation chamber 116 to 15 the seed collector 120 where each seed is removed from the face of the separating Wheel 112. After each seed is removed from the separating wheel 112, each seed is tunneled, via the seed collector 120, to a seed transfer tube 124, Each seed is then passed through the seed transfer tube 124, via gravity, vacuum or forced air, and transferred to a respective one of orientation pedestals 10 204 of the seed orientation subsystem 200. in various embodiments, the seed collector 120 includes a stripping plate (not shown) that is located in substantially Hush with the face of the separating wheel 112 and is structured to contact and physically dislodge each seed from the respective recessed port as the separating wheel 112 incrementally rotates 25 past the seed collector 120, Thereafter, each dislodged seed is tunneled through the seed transfer tube 124 to the respective one of orientation pedestals 204. Alternatively, in various other embodiments, each seed can be released from its respective recessed port by temporarily terminating the vacuum at each individual recessed port as the individual moused port is positioned adjacent the seed collector 120: Thereafter, the dislodged seed is transferred to the 'respective one of orientation pedestals: 204, via the transfer tube 124. In still other embodiments, each seed can be blown from the respective recessed port $ by temporarily providing forced air at each individual recessed port as the Individual recessed port is positioned adjacent the seed collector 120. Thereafter, foe dislodged seed is transferred to foe respective oneof orientation pedestals 204, via the transfer tube 124. 2014200607 05 Feb 2014

Additionally, in various embodiments foe seed loading station 100 can 10 include a bulk seed hopper (not shown) having a shaped surface and a vibrating feeder mechanism (not shown) mounted to system support structure above foe seed bin 110. Large amounts of seed can be placed in foe bulk seed hopper where the seed is tunneled onto the vibrating feed mechanism. The vibrating feeder mechanism can be controlled, via the master control system 700, to meter 15 seeds into the seed bin 110 where the seeds are singuiated and transferred to the seed orientation subsystem 200, as described above.

As described; above, in various embodiments, the seed orientation subsystem 200 includes foe plurality of the orientation pedestals 204 and the one or more imaging devices 208. Aridipnaify, the seed orientation subsystem 200 20 can include a seed pick and place device bank 212 movabiy mounted to stationary system support structure of the seed sampler system 10.

Referring now to Figures 2A, 3 and 4, in various embodiments, each orientation pedestal 204 can include a seed seat 216 coupled to a distalend of a shaft 220 that is rotatable by motor 224, as controlled by the master control 25 system 700. The seed seats 216 are structured to receive seeds from the seed transfer tube 124 and retain the seeds for imaging by the imaging deviee(s) 208, as described below. For example, in various embodiments, a fade 226, i.e.,8 top surface, of each seed seat 216 can include a recessed seed reservoir 228, e.g., ~i4- 2014200607 05 Feb 2014 a groove, channel or concavity, structured to receive seeds tom the seed transfer tube 124 and retain the seeds tor imaging by the imaging device(s) 208, as described below. Moreover, tie seed reservoirs 228 can be formed to have a shape that wilt cause each seed to tie Tlat’ within the respective seed reservoir 5 228. That is, each seed reservoir 228 can be ; contoured id cause each respective seed to He within the reservoir 228 on one of the opposing broad sides of the respective seed, Therefore, each seed lies bn one of the respective opposing broad sides such that the germ of the Sded is viewable by the imagihg^ device(s) 208 and the tip’ of each seed is pointing anywhere within a 3604 plane 10 that is substantially orthogonal to the respective orientation pedestal shaft 220, Additionally, in various embodiments, each orientation pedestal 204 can be communicatively connected to avacuum source fnot shown) that is controiied by the master control system 700 to selectively provide a vacuum at the face 226 and/or seed reservoir 228 of each seed seat 216. Therefore, in such .1$ embodiments, when a seed Is deposited onto an orientation pedestal 204, as described below, the seed is placed on the seed seat 216 of the respective orientation pedestals 204, and a vacuum can be provided at the facto 226 and/or seed reservoir 228 of the respective seed seat 218 to firmly arid steadily retain the seed thereon. Additionally, in various implementations, each seed seat 216 20 and/or seed reservoir 228 can be sized, and fabricated from a suitable friable materia!, such that when the vacuum is provided, each seed is firmly retained ori the respective orientation pedestal 204 without damaging the seeds or compromising the integrity and germination viability of the seeds, e.g., without damaging the seed embryos. 25 Referring now to Figure 2B, in various embodiments, each orientation pedestal 204 can include a vaeuum ftozzie 252 connected to toe distal end of toe shaft 220 that is rotatable by the motor 224. Each vacuum nozzle 252 is communicatively connected id a vacuum source (not shown) that is borttroiled by -15- 2014200607 05 Feb 2014 the master control system 700 to selectively provide a vacuum at a tip 256 of each vacuum nozzle 252* Therefore* In such embodiments, when a seed is deposited onto ah orientation pedesta! 204, as described below, the seed is placed on the tip 256 of the respective orientation pedestals 204, and a vacuum 5 is provided at the tip 256 to firmly and steadily retain the seed thereon, Moreover, the tip 256 of each vacuum nozzle 252 is Structured Such that each seed Is securely retained on the tip 256 of a respective vacuum nozzle 252 having the Tip* of each seed pointing anywhere within a 360° plane that is substantially orthogonal to the respective orientation pedestai shaft 220, 10 Additionally, in various implementations, each vacuum nozzle 252 amt tip 256 can be sized* and fabricated from a suitable pliable material, such that when the vacuum is provided* each seed is finely retained on the respective orientation pedistal 204 without damaging the seeds or compromising the integrity and germination viability of the seeds, e.g., without damaging the seed embryos. 15 Referring now to Figure 2C, in various embodiments, each orientation pedestal 204 can include a seed retention daw 260 connected to the distal end of the shaft 220 that is rotatable by the motor 224, Each seed retention daw 260 includes a plurality of gripping fingers 264 that alb: controllable by the master contfoi system ?00: to grasp and retain seeds delivered to the respective 20 orientation pedestai 204 by the seed loading;: station 100, Therefore, in such embodiments, when a seed is transferred to an orientation pedestal 204, as described beldw, the seed is grasps and firmly retained by the respective seed retention daw 260, as contruilbd by the master control system 700i Moreover, the each seed is securely retained by the respective seed retention claw 260 25 having the Tip’ of each seed pointing anywhere within a 360° plane that Is substantially orthogonal to the respective orientation pedestal shaft 220. Addltldnaliy, It various implementations, the master control system 700 controls the pressure exerted on each seed by the respective gripping fingers 264 such that each seed is firmly retained by each respective seed retention ciaw 260 without damaging the seeds or compromising the integrity and germination viability of the seeds, e^g.. without damaging the seed embryos. 2014200607 05 Feb 2014

Referring now to Figure 2D, in various embodiments, each orientation 5 pedestal 204 can include a micro needle 268 connected to the distal end of the shaft 220 that is rotatable by the motor 224. in such embodimehts, when a seed; is transferee to an orientation pedestal 204, as described below, the seed is stuck or Impaled by the respective micro needle 268 to retain the seed thereon, Moreover, the each seed is securely retained on the respective micro needie 288 10 having the lip’ of each seed poindhg anywhere within a 360e plane that is substantially orthogonal to the respective orientation pedestal shaft 220. Furthermore, by virtue of the very smaiidiameter, e.g., 0.02 mm to 0.07 mm, of each micro needle 268 each seed is firmiy retained one each respective micro needle 268 without damaging the seeds or compromising lie integrity and 15 germination viability of the seeds, e.g., without damaging the seed embryos.

Referring now to Figure 2E, in various embodiments, each orientation pedestal 204 can include an air dispereioo head 222 connected to the distal end of the shaft 220 that is rotatabie by the motor 224. Each air dispersion head 272 is Oommunicatively connected to an air sourcefnot shown) that Is controlled by 20 the master control system 700 to selectively provide a flow of air at a face 276 of each air dispersion head 272. Theface 276 of each air dispersion head 272 includes a pturafity of onflees (not shown) such that the flow of air can be dispersed across each respective face 276; More particularly, in such embodiments, when a seed is transferred to an orientation pedestal 204 from the 25 seed loading station 100, as described below, the flow of air dispersed across the face 276 of the respective air dispersion head 272 provides a cushion of air that slightly levitates each respective seed and steadily retains each respective seed above the respective air dispersion head 272; Moreover, each air dispersion -17- 2014200607 05 Feb 2014 head 21¾ retains each seed such that the ‘tip’ of each seed is pointing anywhere Within a 360° plane that is substantially orthogonal to the respective orientation pedestal shaft 220, Additiphaliy, the cushion of air provided at each air dispersion head 272 firmly retains each respective seed on the respective 5 orientation pedestal 204 without damaging the seeds or compromising the integrity and germination viabtiity of the seeds, e.g., without damaging the seed embryos.

As best shown in Figures 2 and 4, in various embodiments, the seed singuiator 104 is siidabiy mounted to a linear stage 108 such that the seed 10 singuiator 104 can be translated along a length of the linear stage 108 to selectively position a dispending end 128 of the seed transfer tube 124 above tie seed seat 21© of each orientation pedestal 204. in varieus impierrsentations, the linear stage 108 induces a translating track 132 and an actuator 138 operabie to bidirectionally mpve a carriage 138, to which the seed singuiator 104 is mounted, !5 along the length of the translating track 132, The master control system TOO is operabie to control and coordinated the operations of the seed singuiator 104, as described above, and the movement of the seed singufator 104 along the linear stage 108 to sequentially deposit a single seed onto the seed seat 216 of each orientation pedestal 204, via the seed transfer tube 124. 20 Referring now to Figure 4A, in various embodiments, the seed singuiator 104 is statipnariiy mounted ip System support striiCture and the seed loading station 100 includes a mtary diverter assembly 140. The rotary diverter assembly 140 is controllable by the master control system 700 to sequentially divert each seed slnguiaibd by seed singuiator 104 into one of a plurality of seed 25 transfer tubes 124. A proximal end 142 of each transfer tube 124 is connected to the a diverter block 144 of the diverter assembly 140, and a distal end 148 of each transfer tube is stationariiy positioned above a corresponding one of the orientation pedestals 204. Mote particularly, the diverter assembly 140

-IS 2014200607 05 Feb 2014

IS

IS a^diiic>r>siiy ineludes a rotary diverter head 148 that: is controllable by the master control system 700 and a rotary motor 154, e.g., a servomotor, to direct each respective singuiated seed into one of a plurality of diverting passages 150 extending thrpogh the diverter block 144. The diverter head 148 is rotafionaily connected to the seed coiiector 120 via a rotatable collar 152. Thus, as a seed is removed from the: separating wheel 112, as described above, the seed travels through the seed coiiector 120 and the rotatable collar 152 into an internal ehannei (not shown) of the diverter head 148,. As; each seed is singuiated, the master control system 700 incrementally rotates the diverter 148, via the motor 154, about a longitudinal axis of the rotatable coilar 152 to sequentially position a dispensing end of the diverter head internal chahhel in coaxla! alignment wild each of tee diverting passages 150, Accordingly, each Singylated seed wi be diverted to a corresponding one of the seed transfer tubes, and thereby deposited onto a corresponding one of the orientation pedestals 204.

Referring now to Figures 3 and 4, the imaging device(s) 208 is/are mounted to system support structure above the orientation pedesta!s204 such that each of tee Grientation pedestais 204, more particularly, the seed seat 216 of each of the orientation pedestals 204, IS within the field of view of one or more of the imaging devicefs) 208, Accordingly, as each seed is deposited onto a 20 respective seed seat 216, the seed is placed within the fieid of view of one or more of the imaging device(si 208. Subsequently, the respective imaging deviee(s) 208 acquirers) orientation image data of each seed that is communicated to the master control system 700, The imaging device 208 can be any imaging device suitable for collecting images of the seeds deposited onto 25 the orientation pedestals 204. For example, in various embodiments, tee imaging device 208 can comprise a high speed, high resolution digital camera, such as a disruptive visual technology (DVT) machine vision camera for coiiecting visual digital image data of each seed., Alternatively, tee image data ~i0- 2014200607 05 Feb 2014 OTlSeqied can be near infra-red (NIR) image data, NMR/MRl image data, or any other type image data.

As descried above, when a seed is deposited onto the orientation pedestal 204, each seed is oriented such that the ‘tip' of each seed is pointing 5 anywhere within a 360° plane that is substantially orthogonal tothe respective orientation pedestai shaft 220, Trie image data collected is communicated to the master control system 700, where upon execution of an orientation algorithm, the image data is analyzed to determine the orientation of each seed, e.g, .which direction the ‘tip’ and/or the ‘Grown’ of each seed is pointing as it sets on the IQ respective orientation pedestalseat 216. For example, in various embodiments, execution of the orientation algorithm, e.g., a vector determining algorithm, can determine a location of a centroid of each seed and identify the· farthest point from the centroid of the respective seed as the seed ‘tip’ and the end of the respective seed opposite the ’tip* as the ’Grown' of the respective seed. 15 Subsequently, based on the determined orientation of each respective seed, the master control system 700 wifi position each seed in a; desired orientation. That is, the master control system 700 will rotate each respective prieritatipri device shaft 220, via the motors 224, ip pfieht each seed such that either the ‘tip1 or the 'Grown' of each seed pointing in 8 desired direction, e,g.t pfthc^pnaiiy toward or 20 orthogonally away from a front edge 308 of the sampling subsystem 300 (shown in Figure 5). Orienting the seeds in the proper position minimizes the impact to the Seed's germination viability whin a sample is removed from the seed, as described below.

Referring now to Figures 3 and 4, in various embodiments, once each 25 seed is positioned in the desired orientation, each seed is moved to me sampling subsystem 300, via the pick and place device bank 212, in various embodiments, the pick arid place device bank 212 includes a plurality of pick and place devices 232, i.e„ a number of pick and place devices 232 equal to the number of orientation devices 204, Similarly, fee sampling subsystem 300 includes a number of sampling retention fixtures 304 equai to the number of orientation devices 204. 2014200607 05 Feb 2014

JO 15 20

Each pick and place device 232 includes at least one seed picker 236 coupled to an actuator arm 240 of a linear arid rotary actuator 244 that is controlled by the master control system/OO. in operation, once each seed is oriented in the desired orientation, as described above, the master control system 700 will operate each respective actuator 244 to position an extractiontip 248 of each seed picker 236 directly above the respective seed retained by the respective seed orientation pedestal 204. The master control system 700 will then control each respective actuator 244 to lower the respective actuator arm 240 and seed picker 236 cdnnectod thet^d such that the extraction tip 248 can grasp the respective seed. Once each seed is grasped, i.e., secured or retained, by toe respective extraction tip 248, the master control system 700 will control each actuator 244 to raise toe respective actuator arm 240 and seed picker :236 connected thereto such that the respective seep is Sifted from the respective sped seat 216. Thereafter, via control by the master control system 700, each actuator 244 moves each respective seed to a position directly above the corresponding sampling retention fixture 304. Subsequently;, toe master control system 700 will control each actuator 244 to lower the respective actuator arm 240 arid seed picker 236 connected thereto such thatthe extraction tip 248 can place each respective seed into the corresponding sampling retention fixture 304, whereby each seed is held and a sample is removed, as described below; Particularly, utilizing orientation data, the master control system 700 will orient each seed on trie respective orientation pedestai204 such; that each seed will be placed into the corresponding sampling retentionfixiure 304 having toe portion of the seeds containing the embryo, i.e., trie 'tip', pointed away from a cutting device 340 associated with each respective sampling retention fixture 304. -21- 2014200607 05 Feb 2014

Accordingiy, when placed intoone si the sampling retention fixtures 304, each seed will be positioned such that the 'Crown'of each seed is pointed toward, or facing, the corresponding cutting device 34. Therefore, each sample will be removed from the 'Crown' of eacto containing the cotyledon 5 and/or endosperm tissue of the seed, as described below, so that the germination viability of each seed is preserved.

The pick and place device extraction tips 252 can be any tip or device that is suitable to grasp, or pick up, the seeds from the seed seats 216 and place each seed into a respective one the sampling subsystem sampling retention 10 fixtures 304 without compromising the integrity and germination viability of the seeds, e.g., without damaging the seed embryos. Tor example, in various embodiments, each extraction tip 248 can comprise a vacuum nozzle wherebya vacuum is utilized to lift and retain each respective seedas the seed Is removed from toe respective seed orientation pedestal 204 and placed in toe 15 corresponding sampling retention fixture 304. Alternatively, each extraction tip 248 can comprise a mechanical daw, a micro needle or any other holding device that wilt not compromise the integ rity and g ermina tion viability ofthe seeds as the seeds are transfemed from the orientation pedestals 204 to the cpfTesppnding sampling retention fixtures 304. Additionally, in various embodiments, the 20 actuators 244 can be operable to rotate toe respective actuator arms 240, either independently or ft unison, to transfer the seeds from the respective seed seats 216 to the corfesponding sampling retention fixtures 304. Aitematively, the pick ; and place device 2T2 is structured and operable to laterally move each pick and place device 232 between the orientation subsystem 200 and toe sampling 25 subsystem 300, either independently or in unison, to transfer toe seeds from the respective seed Seats 21 δ to the corresponding sampling retention fixtures 304.

Attobuijh: the orientation subsystem 200 has been described and Illustrated herein to utilize the pick and place devices 232 to transfer toe seed -22- 2014200607 05 Feb 2014 from the orientation pedestals 204 id trie sampling retention fixtures 304, any suitable mechanism can be utilized to do so and remain within the scope of toe present disclosure. For example, I is envisioned that the orientation subsystem 200 can include any suitable apparatus, device, assembly, etc., structured and 5 operable to rotate, flip, slide or otherwise move each seed from toe orientation pedestals 2p to the sampling retention fixtures 304 such that the top* of each seed faces away from the cutting device 340 associated with each respective sampling retehtldh fixture 304.

Referring how to Figures 2, 3, 4 and 5, in various embodiments, the Ϊ0 sampling subsystem 300 includes a base platform 312 mounted to system support structure and to which the sampling retention fixtures 304 are mounted. The sampling subsystem 300 further includes a plurality of sampling assemblies 313, he., a number of sampling assemblies 316 equal to tod number of sampling retention fixtures 3Q4. 15 Generally, in operation, each seed is heidwithin toe respective sampiing retention fixture 334 and a sample of each seed is removed from theOrown', e g., a portion of each seed containing the cotyledon and/or endosperm tissue, by the corresponding sampiing assembly 316 without damaging toe seeds or pompromising the germination viability or integrity of toe seeds. Each sampled 20 seed is then transferred to the seed collection assembly 500, and each corresponding removed sample Is transferred ίο the sample collection assembly 600, via the seed and sample transport subsystem 400;, as described below.

In various embodiments, each sampiing retention fixture 304 comprises a gripper block 330 structured to slide on a rail ; 324, e.§;, a track or dovetail, 25 between a seed receiving position arid a seed sampling position, as cprifrotled by the master control system 700. in various Impteritoritations, each gripper biock 320 includes a seed channe! 328 and a pair of opposing damping fingers 332. The clamping fingers 332 are movable, as controlled by me master control -23- 2014200607 05 Feb 2014 system 700, between an opened position, in whic^ seeds car» be placed between the clamping fingers 332 by tie respective pick and place device 232, and a ciosed position, in which the clamping fingem332securelyhold each respective seed for sampling by the corresponding sampling assembly 316. 5 Thus, so various embodiments, the pick and place devices 232 will remove seeds tom the respective onerttatton pedestals 204 and then place the seeds Within the seed channel 328 and between the clamping lingers 332 of the respective gripperbioek320 that: has been moved to the seed receiving position. Subsequently, the master control system 700 wiil control the operation of the 10 gripper blocks 320 to move the damping fingers 332 to the closed position and the gripper blocks 320 to the seed sampling position. It should beunderstood that when each seed has been placed in and retained by the respective gripper block 320, and the gripper blocks 320 have been moved to the seed sampling position, a portion of each seed is sufficiently exposed to allow the corresponding 15 sampling assembly 316 to remove, or extract, a sample from each respective seed. For example, in various embodiments, at portion of the ‘Crown’ of each respective seed is exposed such that a sample can be removed from the ‘Crown' without interference by the respective gripper blocks 320.

Ip various implementations, using the imaging data obtained by the 20 orientation subsystem 200, the sampling position for each gripper block 320 is independently deteirnined by the master oontro! system 700 such that only a desired amount of sample is removed or extracted from each respective seed. That is, the distance each gripping block 320 travels along the respective rail 324 can be independently and dynamically controlled to position each respective 25 seed relative to the corresponding sampling assembly 316 such that only a :desired amount of each seed is remoyed or extracted as the sample.

In various embodiments^ each; sampling assembly 316 indudes a rotary drive motor 336 operable to control the movement; of a cutting device 340 to ♦ 2014200607 05 Feb 2014 remove or extract the sample, e.g., tissue sample, from each respective seed, in various implementations fee cutting devices 340 comprise cutting blades that are connected to shafts .ofthe dnve mot®s 336 sudi that rotation of the shafts will move the respective cutting blades In an arced up-and-down guillotine-like 5 motion to cut the respective seed arid remove fie samples, in various other implementations the cutting blades can be connected to the shafts of the drive motors 336 via a cam device such that rotation of each shaft will move the respechve cutting blade in an up-and-down-and-across slicing motion to remove the respective^ Isifnpies. The cutting blades can comprise any cutting blade 10 suitable to cut the seeds in accordance of the cutting motion imparted by tile respective drive motor 336 without erushing the seeds and damaging fie embryos, thereby compromising the integrity and germination viability of the seeds. For example, in various embodiments, the cutting blade can comprise a blade similar to a utility knife blade or a scalpel. 15:: Once each sample has been removed or extracted from a seed, the respective drive motor 336 will return the respective cutting device 340 to a ready to cut position for the next seed, in vanous implementations, each drive motor 336 heeds only to rotate about 30 degrees to cut the respective seed to remove the sample. 20 As described above, the orientation subsystem 200 utilizes the orientation dfta to orient each seed on the respective orientation pedestals 204 so that each seed is placed into fie corresponding sampling retention fixture 304 having the Crown’ of each seed facing, or pointing toward, the respective cutting device 340, and ‘tip’ of each seed pointing away from the respective cutting devics 340. 25 Therefore, each sample will be removed from the Crown* of each seed so that the respective Seedembryos will not bedamagedand the germination viability of each seed will be preserved. Additionally, as described herein, the master control system 700 spatially positions the ‘Crown* of each seed with regard to •25- 2014200607 05 Feb 2014 each corresponding cutting device 340 such that only a desired amount of the ‘Crown* of each seed is removed to thereby preserve the germination viability of each seed.

Referring now to Figure 6> in various embodiments, each sampling 5 assembly 316 can comprise a linear actuated seed sampling devlce 344 that includes a linear actuator 348 controllable by the master control system 700 to bidirectionally move a drive rod; 352 along a longitudinal axis X of the linear actuator 348. Additionally; in various embodiments, each cutting device 340 can comprise o sample extractlGn bit 356 connected to a distal end of therespective 10 drive rod 352. The sample extraction bit 356 is structured to remove or extract samples from each respective seed, Moreparticulariy, once a seed is retained within a sampling retention fixture 304,the master contro! system 700 can control the operation of the respective linear actuator 348 to move the respective sample extraction bit 356 into contact with the respective seedsuch that tee sample 15 extraction bit 356 removes a sample from the seed.

For example, in various emt^imehtSi the sample extraction bit 356 can be a hollow cutting tube, such as a biopsy punch, cork borer, or Similar device tee will be pushed through each seed, via the linear actuator 348. to perforate each seed and extract the samples. In other embodiments, each linear actuator 20 348 can comprise a rotary linear actuator and each sample extraction bit 356 can comprise a cutting spin bit Accordingly, tee master control system 700 can control the linear actuator 346 to simultaneously rotate, e.g., spin, the cutting spin bit and move the respective sample extraction bit 356 into contact with the respective seed to drill a hole In each seed and extract the samples. 25 Referring now to Figure 7, in various embodiments, each cutting device 340 can comprise a rotary motor 380 connected to the distal end of the respective drive rad 352 a rotary cutting blade 364 connected b a shaft of the respective motor 360, in such embodiments, once a seed is retained within a sampling reienilpn fixture 304, the master control system 700 part control the operation of the respective linear actuator 348 and motor 360 to rotate the cutting blade 364 and move the rotating cutting blade 364 into contact with the respective seed to cut a sample from the seed, 2014200607 05 Feb 2014 5 Each cutting device 340 cancomprise and device satiable to remove or extract a desired amount of eachseed to provide a sample, e.g., a tissue sample, of each seed. For example, in various embodiments, each cutting device MO can comprise an knife, a laser, a water jet, vibrating blade or bit, or any other cutting device Suitable to remove the desired sample each seed, 10 Furthermore, in various embodiments, each cutting device 340 and indude a plurality of cutting blades, extraction bits, rotary cutting blades, air knifes, lasers, water Jets, vibrating bladesorbits, etc,, such that a plurality ot portions of each sped ere removed. For example, in various embodiments, each cutting device can include two cutting blades, extraction bits, rotary cutting 15 blades, etc. in such embodiments, the master control system 70D can eontroi the operation of the sampling assemblies 316 and the sampling retention fixtures 304 to remove and discard a first portion of each seed comprising the skin or coat of each seed, and subsequently or simuitaheduSty remove or extract a second portion of each seed comprising tissue, e.g., cotyledon and/or endosperm, 20 usable as the sample and suitable for genetic analysis, as described below.

Alternatively, in various embodiments, a plurality of portions of each seed can be removed utilizing putting devices comprising a single cutting biade, extraction bit, ratary cutting blade, etc. For example, in such embodiments, the master control system 700 can control the operation of the sampling assemblies 25 316 and the sampling retention fixtures 304 to remove and discard a first portion of each seed, thereby removing and discarding a portion of the skin or coat of each seed. Subsequently, the master control system 700 can eontroi the operation of the sampling assemblies 316 and the sampling retention fixtures 3Q4 2014200607 05 Feb 2014 -27- to remove or extract a second portion each seed comprising tissue, e.g., cotyledon and/or endosperm, usable as the sample suitable forgenetic analysis, as described f^low.

Additionally, in various embodiments, the master control system 700 can 5 control the operation of the sampling assemblies: 316 and the sampling retention fixtures 304 to remove different sample sizes or amounts from each respective seed. That is, the master control system 700 can position the ‘Crown* of each seed any partieuiariy distance from the respective cutting device 340 so that any a desired amount of size of sample can be removed, orextracted. 10 it should be understood that each sampling retention fixture 304 is structured and operable to firmly retain each respective seed such that each respective seed does not move and is not damaged or the germination viability of each respective seed compromised as the corresponding sampling assembly 316 remove? a sample tpm each seed. For exarnple.with exemplary reference 15 to pigum p, in various embodiments, each clamping finger 332 can be fabricated from; or include a layer of, pliable material such as rubber or other synthetic rubber-like material feat will aid in firmly and securely holding each respective seed between the clamping fingers 332 without damaging fee seed embryos and compromising the germination viability or integrity of the seeds, e.g., without 20 damagingihe seed embryos.

Referring now to Figure 8A, in various embodiments, each sampling retention fixture 304 can comprise a pair of opposing pliable Piddles 362 coupled to actuators 365 mounted dri a base 366. In such embodiments, as each seed is transferred from the orientation subsystem 300 and placed between the pliable 25 paddles 3621 the master control; system 700 controls the actuators 365 to firmly grasp and hold each respective seedlbetween pliable paddles 362. The pliable paddles 362 can be fabricated from any pliable material: Such as rubber or other synthetic rubber-tike materia! feat will aid in firmly and securely holding each -28- 2014200607 05 Feb 2014 respective seed between the pliable paddies 362 without damaging the seeds or compromising the germination viability or integrity of the seeds.

Reciting now to Figure 8B, in various other embodiments, each sampling retention fixture 304 can comprise a grooved base 368 including a recessed 5 seed channel 369 and a pliabie hood 370 pivotaliy connected to the base 368 via a rotary actuator 372. in such embodiments, as each seed is transferred from the orientation subsystem 300 and placed into the seed channel 369, the master control system 7Ό0 controls the rotary actuator 372 to position die pliable hood 370 in contact with each respective seed, thereby firmly holding each respective 10 seed within the respective seed channel 369, The pliable hood 370 can be fabricated tom any pliable materia! such as rubber or other synthetic rubber-like material that will aid in firmly and securely holding each respective seed within the seed channel 369 without damaging the seeds or compromising the germination viability or integrity of the seeds. 15 Referring now to Figure 8C, in various embodiments, each sampling retention fixture 304 can comprise a pair of opposing grooved paddles 374 coupled to actuators 376 mounted on a base 378. in such embodiments, as each seed is transferred from the pnentaiidh subsystem 300 and placed between the grooved paddles 374, the master control system 700 controls the actuators: 20 376 to firmly grasp and hold each respective seed between grooved paddies 374, Accordingly, each respective seed will be firmly and securely held within the grooves of toe grooved paddies 374 without damaging toe seeds or compromising the germination viability or integrity of the seeds.

Referring now to Figure 8D, in various other embodiments, each sampling 25 retention fixture 304 can comprise a base 330 including and a vacuum cup 382 mounted to a distal end Of a drive rod 384 of a linear actuator 335. in such embodiments, as each seed is transferred from the orientation subsystem 300 and placed oh the: base 380, the master control system 700 controls the linear 2014200607 05 Feb 2014 -29 actuator 385 to position the vadium cup 382 in contact with each respective seed. A vacuum is then provided at the respective vacuum cup 382. Accordingly, each respective seed wi be tirmiy and securely held within the respective vacuum cup 382 and on toe respective base 380 without damaging 5 the seeds or compromising the germinatiph viability or integrity of the seeds.

Referring now to Figures 5, 8E and 8F, In various embodiments, each sampling reteutigp fixture 304 can comprise a clamping block 388 fixedly mounted to toe base platform 312 and including a base 392 and a cover 394 for retaining seeds during sampling. Additionally, each sampling retention fixture ID 394 can include a datum block 390 slitiihgiy mounted to the base platform 312 tost is structured and operable to align each seed within the respective damping block such toat a particular desired amount of each seed Is removed during sampling.. A top surface Of each clamping block base 932 includes a first tail portion of a seed nest 398, a bottom surface of each respective damping 15 biock cover 394 ihcfbties a second tail portion 3968 of the respective seed nest 398, and a top surface of each datum biock 390 includes a head portion 396Bof the respective seed nest 398. In such embodiments, each damping block cover is inliaiiy placed in an ‘Opened’ position, as controlled by the master control system 700, providing access to the first tali portion of 398A of the respective 20 seed nest 398. Additionally, and each datum block 390 Is initially placed in a ‘Sampling’ position, as controlled by the master cpntroi system 700, wherein each datum block 390 is placed in close proximity to the respective damping block base 392. When each datum block 390 is placed in the 'Sampling1 position, a cutting gap 398 is provided between the respective clamping block 25: base 392 and the datum biock 390, and each datum block 390 substantially covers the respective extraction aperture 388,

With the damping biock cover 394 of any sampling retention fixture 304 in the Opened’ position and the datum block 390 in the ‘Sampling' position a seed -30- 2014200607 05 Feb 2014 can be transferred from the orientation subsystem :300 and placed into tie respective seed nest 390, i.e., the first tail portion 396A and the head portion 396C of the respective seed he$t 396. More particularly, each seed is placed into a respective seed nest 396 such that the ‘Crown' of each seed abuts a distal 5 end 399 of the respective seed nest head portion 396G, thereby .consistently and accurately aligning the ‘Crown’ of each seed relative to the cutting gap 396. Accordingly, each cutting device 349 will consistently and accurately remove only a specific sample amount of each seed, as described below.

Once a seed has been pieced into the respective seed nest 396, i.e., tie 10 first tail portion 396A and the head portion 3960 of the respective seed nest 396, the master control system 700 commands fee respective clamping block cover 394 to a 'Closed’ position, whereby each respective seed is firmly retained within the seed nest 396 without damaging the seeds or compromising the germination viability or integrity of the seeds, 'i.e., without damaging the seed embryos. IS Furthermore, in such embodiments, the cutting device 349 can comprises a rotary cutting blade 364 strectured and operable, via an associated motor (not shown) and control of the master control system 700, to rotate and bidirectionally move vertically and/or horizontally with respect to me clamping: block 366 to remove each respective sample; More particularly, the rotary cutting blade 364 20 can be structured and operable to rotate at a high velocity and traverse through each respedtive seed along the pathway provided by the cutting gap 396,

Once a seed has been sampled,: the respective datum block 390 is moved, as controlled by the master control system 700, away from the cdrrespdnding clamping block base 392, to an 'Extraction" position, thereby 25 uncovering the respective extraction aperture 386 and creating an extraction gap 395 between the damping block base 392 and the datum block 390, The seed sample is then extracted, via gravity, vacuum and/or forced air, from the head portion 396C of the seed nest 396 through the extraction aperture 386 and 2014200607 05 Feb 2014 deposited into ope of tie sample collection trays 604, e.g., microplates, as desctebed below. Subsequently, the master control system 700 commands the clamping block cover 394 to tie ‘Opened' position allowing the sampled seed to be extracted via gravity, vacuum and/or forced air, from the first tail portion 396A 5 Of the seed nest 396 through the extraciionaperture386 and to be deposited into one of the seed collection trays 504, as described below.

In various implementations, the damping block cover 394 can be fabricated from, or include a liner fabricated tom, a pliable material such as rubber or other synthetic rubber-like material that will aid in firmly and securely to holding each respective seed within the seed hast 396 without damaging toe seeds or compromising the germination viability or integrity of the seeds. Alternatively, in other implementations, toe damping block cover 394 can comprise a piiabie fat cover, i.e., absent the second toil portion 396B of the seed nest 396, such as that described above with regard to Figure 8B. 15 Moreover, it is envisioned that any combination of the sampling retention fixture 304 embodimehts described above with reference to Figures8A, 8B, 8C, 81¾ BE and 8F can bp utilised to fiitniy and securely hoto each seed during sampling without damaging toe seedsor^ compromising toe germination viabiiity Or integrity of the seeds.. 20 Referring now to Figure 2, each seed collection tray 504 includes a plurality of seed wells 506, each of which are adapted for receiving a seed dispensed from the seed and sample transport subsystem 400, as described beiow. For example, in various embodiments, each seed collection tray 504 can Include twenty-four wells 506, Simiiarty.each sample collection tray 604 inc!udes 25 a plurality of sample welis 606, each of which are adapted tor receiving a sample dispensed from the seed and sample transport subsystem 460, as described below. For example, in various embodiments, each sampie coitection tray 604 can comprise a mictopiate iriciuding ninety-six mlcro weils 606. 2014200607 05 Feb 2014 -32-

Once a seed his been sampled, the seed and sample are deposited into the seed collection assembly 500 and the sample cofieetiqn assembly 600, respectively, via the seed and sample transport subsystem 400, Moreover, the seed and sample transport subsystem 400 is controllable by the master control 5 system 700 to sort the sampled seeds to the seed collection trays 504 and sort the corresponding seed sample to the sample collection trays 604 while tracking and mapping the locations of the corresponding sampled seeds and seed samples in the respective seed and sampie collection trays 504 and 604.

As described above, in various embodiments, the seed and sample 10 transport subsystem 400 includes a plurality of diverter assemblies 404. More particularly, the seed and sample transport subsystem 400 includes a number of diverter assemblies 404 equal to the number of sampling assemblies 316. Moreover each diverter assembly 404 is mounted, via system support structure, adjacent a corresponding one of the sampling assemblies 316 such that as each 15 sampling assembly 316 removes a sampie from a seed, the sampie and sampled seed are sequentially deposited into the respective diverter assembly 404.

The seed and sample transport subsystem 400 additionally includes a number seed deposit tubes 403 equal to the number of diverter assemblies 404, Each : seed deposit tube 408 includes a proximal end connected to a respective 20 one of the diverter assemblies 404 and an opposing distal end positioned above a seed tfay piailbrfn 503 of the seed collection assembly 500, The seed tray platform 508 is structured to removable retain a plurality of The seed collection trays 504 in fixed positions and orientations. Furthermore, the seed and sample transport subsystem 400 includes a number of sample deposit tubes 4l t equal 25 to the number of diverter assemblies 404. Each sample deposit tube 412 includes a preximal :end connected to a respective one of the diverter assemblies 404 and an Opposing distal end positioned above a sample fray platform 608 of the sample collection assembly 600. The sample tray platform 608 is structured -33- 2014200607 05 Feb 2014 fo removable retain a plurality of the sample collection trays 604 in fixed positions and orientations.

Each diverter assembly 404 is structured and operable, via control by the master control system 700, to divert seed samples into the corresponding sample 5 deposit tube 412, and divert the corresponding sampled seeds into the seed deposit tube 408. Generally, as a seed is sampled, the respective sample is extracted, vis gravity, vacuum andfor forced air into the corresponding diverter assembly 404, whereby the master controller 700 commands toe divertor assembly 404 to dived the Sample into the respective sample deposit tube 412. to The respective sample is then transferred, via gravity, vacuum and/or forced air, through the sample deposit tube 412 and is deposited into a selected one of the wells 608 of a selected one of the sample qolleetton trays 604 removably mounted to the sample tray platform 608. Similarly, the respective sampled seed Is subsequently extracted, via gravity^ vacuum and/or forced air into the IS corresponding diverter assembly 404, whereby the master controller 700 commands the diverter assembly 404 tg divert toe sampled seed into the respective seed deposit tube 408. The respective sampled seed is then ttonsfetred, via gravityi vacuum and/or forced air, through the seed :deposit tube 408 and is deposited into a selected one of the welts 506 of a selected oneof the 20 of seed collection trays 504 removably mounted to the seed tray platform 508,

For example, with reference to Figures 3 and 8, in various embodiments, the sampling subsystem base platform 312 can include a plurality of extraction apertures 386, wberein each extraction aperture 386 is located directly beneath a corresponding one of the cutting devices 340. Additionally, an inlet tube 416 of 25 each diverter assembly 404 Is connectedto:¾ bottom of toe sampling subsystem base platform 312 at a correspondlhg one of the extraction apertures 388. Therefore, as a seed is sampled, the respective sample is extracted, via gravity, yaCuum and/or forced air through the respective extraction aperture 366 and into 2014200607 05 Feb 2014 -34” the corresponding diverter assembly 404, via the respective inlet tube 416- The extracted sample Is then diverted intofoe respective sample deposit tube 412; via tile diverter assembly 404, and deposited into a particular one of the weiis 606 of a partfeular one of the sample collection trays 604. Subsequently, the 5 corresponding sampled seed is extracted, via gravity, vacuum and/or forced air through the extraction aperture 386 and into the diverter assembly 404, via the inlet tube 416. The extracted sampled seed is then diverted into the sample deposit tube 412, via the diverter assembly 404, and deposited into a particular one of the weiis 506 erf a particular one of the seed collection trays 504. 10 To deposit each sample into a particular one of the weiis 606 of a particular one of the sample collection trays 604, the sample tray platform 608 is mounted to a sample tray platform X-Y stage 612 that is a two-dimensional tran|latidh mechanism, including an X axis translating track 616 and a Y axis translating track 618. The sample fray platform X-Y stage 612 additionally 15 includes a first linear actuator 620 operable to bidirectionally move a first carriage (not shown) along the length of the X axis translating track 616, and a second linear actuator 624 operable to bidtrectibnaiiy move a second carriage (not Shown) along the: length of the Y axis translating track 618. The Y axis translating hack 618 is mounted to the first carriage and tee sampie tray platform 20 808 is mounted to the second carriage; Additionally, the sampie deposit tubes 412 are mounted, via system support structure, to position the distal ends above the sampie tray platform X-Y stage 612 and tee sampie tray piatform 608 holding the plu ral ity of sampie collection trays 604.

As each sampie collection tray 604 is placed on the sample tray platform 25 608, a sample collection tray identification number, e g., a bar code, tor each sample eoiiection tray 604 and the location of each respective sampie collection tray 604 on the sample tray platform 608 is recorded in the master control system 700. Based on the recorded location of each sampie eoiiection tray 604 and the 2014200607 05 Feb 2014 -35- known number and configuration of wells 606 in each sample collection tray 604, the master control system 700 determines an X-Y coordinate location of each sample collection tray well 606 on the sample tray platform 608.

In coordination with the sampling of the seeds, the first and second linear 5 actuators 620 and 624 of the sample fray platform X-Y stage 612 are controlled by the master control system TOO to move the sample tray platform 608 within an X-Y coordinate system ip precisely position any one or more selected wells 606 of any one or more selected sample collection trays 604 at one or more target locations directly beneath the distal ends of any one or more of the sample Jo deposit tubes 412. Therefore, each sample wilt be deposited into a particular, selected well 606 of the sample collection sample trays 604.

Bor example, in various embodiments, prior to the samplingsubsystem 300 extracting samples from toe raspective seeds, as described above, toe master control system TOO controls the sample tray platform X-Y stage 612 to 15 position a selected group of wells 606 at the respective target locations beneath two or more of the sample deposit tubes 412. When the selected group of wells 606 is placed at the respective target locations, toe distal ends of each sample deposit tube 412 is in alignmentwith and in close proximityto, or in contact with, a con^pprsding one of toe wells 606 in toe selected group. Subsequently, toe 20 master control system 700 commands the sampling subsystem 300 to extract the samples from the respective seeds. Each sample is then drawn into a corresponding diverter assembly 404 and transferred through toe respective sample deposit tubes 412 to toe corresponding selected sample collection tray well 606, 25 The mastor control system 700 then compares the X-Y location of each deposited extracted sample with the recorded positions of toe wells 606 to map each extracted sample to toe respective well 606 of toe respective sample collection tray 604 in which each sample was deposited. 2014200607 05 Feb 2014

Similarly, to deposit each sampled seed into a particular one of tie wells 606 of a particular one of the seed collection trays 604, the seed tray platform 508is mounted to a seed tray platform X-Y stage 512 that is a two-dimensional translation mechanism, induding an X axis translating track 616 and a V axis 5 translating track 518, The seed hey platform X-Y stage 512 additionally includes a first linear actuator 520 operable to bidirectionally move a first carnage (not shewn) along the length of the X axis translafing track 516, and a second linear actuator 624 operable to bidirectionally movea second carriage (not shown) along foe length of the Y axis translating track 518. The Y axis translating track 10 518 is mounted to the first carnage and the seed tray platform 508 is mounted to the second carriage. Additionally, the seed deposit tubes 408 are mounted, via systemsupport structure, toposfiton the distai ends above the seed tray platform X-Y stage 512 and the seed fray platform 508 holding the plurality of seed coilectidh trays 504, 15 As each seed coilectidh tray 504 is placed on thi seed tray platform 508, a seed collection tray identification number, e,g„ a bar code, for each seed collection tray 504 and the location of each respective seed co! lection tray 5G4 on the seed tray platform 508 is recorded in the master control system 700. Based on the re corded location of each seed collection fray 504 and the known number 20 and configuration of wells 506 in each seed collection tray 504, the master tfonifql system 700 determines an X-Y coordinate location of each seed collection fray well 506 on the seed tray platform 508:,

In coordination with the sampling of the seeds, the first and second linear actuators 620 and 524 of the seed tray platform X-Y stage 512 are controlled by 25 the master control system 70Q to move the seed tray platform 508 within an X-Y coordinate system to precisely position any one or more selected wells 506 of any One or more selected seed colSectidri trays 504 at one or more target locations directly beneath the distal ends of any one of rnore of the seed: deposit 2014200607 05 Feb 2014 -37- tubes 408, Therefore, each sampled seed will be deposit into a particular, Selected well 506 of the seed collection trays 504.

For example, in wanpus embbdimehis, prior to the sampling subsystem 300 extracting samples from tie respective seeds, as described above, the 5 master control system 700 controls the seed tray platform X-Y stage 512 to position a selected group of wells 506 at the respective target iocations beneati two or more of tie seed deposit tubes 408. When the selected group of wells 506 is placed at the respective target locations, the distal ends of each seed deposit tube 408 is in alignment with and in close proximity to, or in contact with, 10 a corresponding one of the wells 506 in the selected group Subsequently, the master control system 700 commands^the sampling subsystem 300 to extract the samples from the respective seeds. Each sampled seed is then drawn into a corresponding diverter assembly 404 and tronsferrortthrough the respective seed deposit tubes 408 to tie corresponding selected seed collection tray well 15 506,

The master control system 700 can then compare the X-Y location of each deposited extracted sampled seed with the recorded positions of the wells 506 to map each extracted samptedi seed to the respective well 506 of the respective seed collection tray 504 in which each sampled seed was deposited, 20 iiore particularly, the master control system 700 can then correlate, link, tie or map the specific well 606 and sample collection tray 604 into which each sample was deposited with the specific well 506 and seed collection tray 504 Into which each corresponding sampled seed was deposited. Therefore, each sample and corresponding seed from which the sample was extras can be 25 tracked for crop analytics, or biher purposes^ For example, breeder can utilize the mapped data linking each seed sample with the respective sampled seed from which it was removed to pre-select seeds and only plant those that contain particuiar genes Of interest. 2014200607 05 Feb 2014 -38-

Although the seed processing system 10 has been described and illustrated herein as including the pick and piece device bank 212 to move oriented seeds from the orientation pedestals 204 to die sampling retention fixtures 304, whereafter the seeds are sariipled, other embodiments of the seed 5 processing system 10 ere envisioned whereby each seed is retained and Sampied on the respective Orientation pedestals. For example, in various embodiments, each orientation pedestal 204 can include any retention fixture 304 described above connected to the distal end of the shaft220 that is rotatable by the motor 224. Therefore, in such embodiments, when a seed is deposited to onto an orientation pedestal 204, the seed is deposited direct on tie respective retention fixture 304 from the seed transfer tube{s) 124. Thereafter, the respective retention fixture 304 firmiy and steadily retains the seed and the seed ^ orierifed with the ’Crown’ toward the respective sampling assembly 316, via toe qrlentipri data collected bytheimaging device(s) 208, as described above. 15 In such embodiments* each of the sampling assemblies 316 are movable, as controlled by toe master contro! system 700, to position the respective cutting device in spatial relationship with each respective seed to remove the desired amount of sample from each seed. Therefore, once each seed has been deposited onto the respective orientation pedestal/reteotson fixture 204/304, each 20 seed is oriented to position the 'Crown’ toward the respective sampling assembly 316. Each respective sampling assembly is then moved to property position the respective cutting device 340 with respect: to toe respective seed ‘Crown', and the sample is removed . The sample and sampied seed are then extracted to the respective diverter assembly 404 and deposited into the corresponding sample 25 and seed collection trays 604 and 504, as described above.

Referring to Figure 9, as used herein* the master control system 700 can comprise a single computer based system, or a plurality of computer based subsystems networked together to coordinate toe operations of the seed 2014200607 05 Feb 2014 -59- proce^ing system 10, as described herein. For example* in various embodiments, the master control system 700can include a plurality of controller subsystems, e,g.,a controller subsystem for each station, system and subsystem of the seed sampler system 10 described herein, in such embodiments, each 5 controller subsystem can include one or more processors or microprocessors that communicate with various seed sampling system sensors, devices, mechanisms, motors, tools, etc., and are networked together with a main controller system to cooperatively operate ail the stations, systems and subsystems of the seed processing system 10. 10 Or, alternatively, the master control system 700 can comprise a single computet based system communicatively connected to ail file various sensors, devices, mechanisms, motors, tools, etc., of the seed processing system 10 to cooperatively operate all the stations, systems and subsystems of the seed processing system 10, For example, in various embodiments, the master contra! 1.5 system 700 is a computer based system that generally includes at least one processor 704 suitable to execute all functions of the master; control system 700 to automatically, or robotically, control the operation of the seed processing system 10, as described herein. The master control system 700 additionally includes at least one electronic storage device 708 that comprises a computer 20 readable medium, such as a hard drive Of any other electronic data storage device tor storing such things as software packages or programs, algorithms and digital intormation, data, look-up tabies, electronic spreadsheets and databases, etc. In various embodiments, toe master contobi system 700 can further include a display 712 for displaying such things as jnlbrmation, data and/or graphical 25 representations, and at least one user interface device 716, such as a keyboard, mousey stylus, scanner and/or an interactive touch-screen on the display 712. In various embodiments the master control system 700 can further include a removable media reader 720 for reading intormation and data from, and/or 2014200607 05 Feb 2014 -40- writing information and: data to, removable electronic storage media Such as floppy disks, compact disks, DVD disks, zip disks, or any other computer readable removable and portable electronic storage media;. Id various impiementahdns, the removable media reader 720 pah be an I/O: port utilized to 5 read external or peripheral memory devices such as thumb drives, memory sticks/cards or externa! hard drives.

Generally, the master control system 700, as described above, includes a seed sampling program, stored oh a storage device, e.g., storage device 708, and executed by a processor, e.g., processor 704, using various inputs from a 10 user interface, e.g., user interface 716, and/or various components, sensors, systems and assemblies Of the peed processing syst^ Particularly, the seed sampling program can include various custom programs, applications, routines, subroutines and/or algorithms that are executable by the processor, e.g., processor 704, to effectuate and control the operation of the seed is processing system 10.

Additionally, in various embodiments, the master control system 700, as described above, can be communicatively connectable to a remote server network, e.g., a local area network (LAN), via a wired or wireless link. Therefore, the master control system 700 can communicate with the remote server network 20 to upload and/or download data, information, algorithms, software programs, etc., and/or receive operational commands. Additionally, in various forms, the master control system 700 can be configured to access the internet to upload and/qr download data, information, algorithms, software programs, etc,, to and from Internet sites and network servers. 25 Samples prepared by the present disclosure can be used for determining a wide variety of physical, morphoSogiciS, Chemical and/or genetic traits. Generally, such traits are determined by analyzing the samples for one or more characteristics indicative of "at least on© genetic or chemical trait. Non-iimiiing 2014200607 05 Feb 2014 -41- examples of characteristics indicative of chemical traits include proteins, oils, carbohydrates, fatty acids, amino acids, biopotymers, pharmaceuticais, starch, fermentable starch, secondaryeompounds, and metabolites. Accordingly, non-limiting examples of chemical traits include amino acid content, protein content, 5 starch content, fermentation yield, fermentation efficiency, energy yield, oil content, determination of protein profiles determination of fatty acid profiles, determination of metabolite profiles, etc.

Non-limiting examples of charaeteristics indicative of genetic baits may include, for example, genetic markers, single nucleotide polymorphisms, simple 10 sequence repeats, restriction fragment length polymorphisms, haplotypes, tag SNPs, alleles of genetic markers, genes, DNA-derived sequences, RNA-derived sequences, prOibOters, 5' untranslated scions ofgenes,3’ untranslated regions of genes, microRNA, siRNA, quantitative trait loci (OIL), satellite markers, transgenes, mRNA, ds mRNA, transcriptional profiles, and methylation patterns. IS The methods arid devices of the present disclosure can be used in a breeding program to seiect plants or seeds having a desired genetic or chemical trait, wherein a desired genetic trait comprises a genotype, a haplotype, an allele, a sequence, a transcript profile, and a methylation pattern. The methods of the present disclosure can be used: in combination with any breeding methodology 20 and can be used to seiect a single generation or to select multiple generations.

In a particular embodiment, the methods of the present disclosure are used to determine the genetic characteristics of seeds in a marker-assisted breeding program. Such methods aiiow for improved marker-assisted breeding programs wherein nondestructive direct seed sampling can be conducted while 25 maintaining the identity of individual seeds from the seed samplerto the field. As a result, the marker-assisted breeding program results in a “high-throughpuf and more efficient platform wherein a population of seeds having a desired trait, 2014200607 05 Feb 2014 -42- marker or genotype can be more effectively buiked in a shorter period of time, with less field and labor resourcespaired;: Such advantages will be more fully descnbed below: in various embodiments, the present disclosure provides a method for 5 analyzing individual seeds within a population of seeds having genetic differences. The method comprises removing a sample comprising cells with nucleic acids from seeds in the jfopulatioh without affecting the germination viability of the seeds; analyzing the nucleic acids extracted from the sample for the presence or absence of at leastsone genetic marker; selecting seeds tom the ϊβ population based upon the results of the riucieic acid analysis; and cultivating plants or plant tissue from the selected seed.

The extracted nucleic acids are analyzed for the presence or absence Of a suitable genetic polymorphism. A wide variety of genetic markers for the analysis of genetic polymorphisms are available and known to those of skill in the IS art. As used herein, genetic markers include, but are not limited to, simple sequence repeats (SSRs), single nucleotide polymorphisms (SNPs), insertions dr deletions (tndeis^ single feature polymorphisms (SFPs, for example, as described in Borevitz et ai. 2003 Gen. Res. 13:513-523) or transcriptional profiles, and nucleic acid sequences. A nucleic acid analysis for the presence or 20 absence of the genetic marker can be used for the selection of seeds in a breeding population- The analysis may be used to select for genes, GIL, alleles, or genomic regions (haplotypes) that comprise or are linked to a genetic marker. Heroin, analysis; methods are known in the art and iheiude, but are not limited to, PCR-based detection methods (for example, TaqMan assays), mlcroarray 25 methods, and nucleic acid sequencing methods. The genes, aileies, QTL, or hapiotypes to be selected for can be identified using newer techniques of molecular biology vdth modifications of ciassicai tweedlng strategies. 2014200607 05 Feb 2014 -43-

As described above, thesampiing systems and methods of this disclosure protect germination viability of the seeds so as to be nondestructive. Germination viability means that a predominant number of sampled seeds, (i.e., greater than 50% of alt sampied seeds) remain viable after sampling. In a 5 particular embodiment, at least about 75% of sampled seeds, and in some embodiments at least about 85% of sampled seeds remain viable. It should be noted mat lovyer rates of germination viability may be tolerable under certain circumstances or for certain applications, for example,, genotyping costs decrease with time beca bse a greaternumber of seeds could be sampied tor me 10 same genotype cost, it should also be noted that sampling does not need to have any effect on viability at all.

In another embodiment, germination viability Is maintained for at least about six months after sampling to ensure mat the sampled seed will be viable until it reaches the field for planting, tn a particular embodiment, the methods of IS the present disclosure further Emprise treating the sampied seeds to maintain germination viability. Such treatment maygeneraliy tndude any means known in the art for protecting a seed from environment in storage or transport; For example, in various embodiments, the sampied seeds may be treated with a poiymer and/or a fungicide to protect the sampled seed while in 20 storage or in transport to the field before planting.

Several methods of preserving single seed identity can be used whiie trahsfefing seed from the sampiing location to the Held, Methods include, but are hot limited to, tmrtsfehihg selected IndiVIduafs to seed tape, a cassette fray, or indexing tray, transplanting with peat pots, and hand-planting from individual 25 seed packets. DMA may be extracted from the sample using any DNA extraction methods known to those of skill in the art which wiii provide sufficient DNA yield, 2014200607 05 Feb 2014 ~44~ DNA.quality, PCR response, and sequencing methods response, A non-limiting example of suitable DNA-extraciion methods Is SOS-based extraction with centrifugation. In addition, the extracted DNA may be amplified after extraction using any amplification method known to those skilled in the art. For example, 5 one suitable amplification method is the GenomiPhi® DNA amplification prep frorn Aroersham Biosciences.

Further, RNA may be extracted lorn the sample using any RNA extraction methods known to those of skill in the art which wiii provide sufficient RNA yield, RNA quality, PCR response, and sequencing methods response, A nondimitihg 10 exampie of suitable RNA-exiraction methods Is SOS-based extraction with centrifugation with consideration tor RNase-free reagents and supplies. In addition, the extracted RNA may be amplified after extraction using any amplification method known to those skilled in die art; For exampie* one suitable amplification method is tie Fuii Spectrum™ RNA Amplification from System IS Biosciences.

Although the present disclosure is generally directed toward the sampling of cucurbit seeds, one skilled in the art would recognize that any seed can generally he utilized in a method or device of the present disclosure, For example, in various embodiments, the seed can be selected from the group 20 consisting of alfalfa seed, apple seed, banana seed, barley seed, been seed, broccoli seed, castorbeah seed, citrus seed, clover seed, coconut seed, coffee seed* maize seed, cotton seed, cucumber seed,· Douglas fir seed, Eucalyptus seed* Loblolly pine seed, linseed seed, melon seed, oat seed, olive seed, palm seed, pea seed, peanut seed, pepper seed, poplar seed, Radiata pine seed, 25 fapeseed seed, rice seed, rye seed* sorghum seed, southern pine seed; soybean SOed, strawberry seed, sugarbeet seed, sugarcane; seed, sunflower seed, sweetgum seed, tea seed* tobacco seed, tomato seed, turf seed* wheat 2014200607 05 Feb 2014 -45- seed, s^^Arabidopmsihafiana seed. In a more particularembodiment, the seed is selected from the group consisting of broccoli seed, cabbage seed, carrot seed, cauliflower seed, Chinese cabbage seed, cucumberseed, dry bean seed, eggplant seed, fennel seed, garden beans seed, gourd seed, leek seed, lettuce 5 seed, melon seed, okra seed, onion seed, pea seed, pepper seed, pumpkin seed, radish seed, spinach seed, squash seed, sweet com seed, tomato seed and watermelon seed. In an even more particular embodiment, the seed comprises cucumber seed, dry bean seed, garden bean seed, gourd seed, melon seed, pea seed, squash seed, sweet com seed, okm seed and 10 watermelon seed.

The methods of the disclosure may further be used in s breeding program for introgressing a trait into a plant Such methods comprise comprising cells with nucleic adds from seeds in a population, analyzing the nucleic acids extracted from each sled for the presence orabsence of at least 15 one genetic marker, selecting seeds from the population based upon die results of the nucleic acids analysis; cultivating a fertile plant from the seed; and utilizing the fertile plant as either a female parent or male parent in a cross With another plant

For example, in various embodiments, the present disclosure provides for 20 improved methods of producing hybrid seed. The method comprises introgressing genetic maie-sieriiity controlled bysingle recessive nuclear genes Into seeds to develop a population of seeds comprising maie-sterile female parents. The population of seeds is then analyzed to identify seeds segregating for the marker; which are selected to be used as female parents to produce 25 hybrid seed. The improved process is advantageous in that it does not require flower removal or hand emasculation, thereby allowing for the use of bees for pollination . Further, large volumes of seeds cap be analyzed by this method and selected in one location and then Shipped anywhere in the world for direct seeding to produce hybrid seed. 2014200607 05 Feb 2014 10

With exemplary reference to melon seeds, five single recessive nuclear genes have been identified in melon for controlling male sterilityi Male-sterile lines always segregate for sterile and fertile plants because they must be maintained by pollinating male-sterile plants (ms/ms) with heterozygous, isogenic male-feiife plants (Svfs/ms). Thus, the use of nuclear genic mafe-ster·!ily requires that foe male-fertile segregants be removed fe>m the female parent rows in hybrid seed production fields. However, by genetically linking a molecular marker to a male-sterile gene, the apparatus, systems and methods of foe present disclosure can be Used to analyze segregating seeds for foe marker and automatically select only seeds predicted to be mafe-stenle.

Two of foe five male sterile (ms) genes in melon have been mapped with molecular markers. The ms3 gene was initially mapped by Park et ai., 2004. 15 (See, Park et a!., “Identification of RAPD and SCAR markers linked to foe rh$3 gene contrdiiing male sterility in meidn (Cucumis melon L.).” J. Amer, Soc. Hort. Sci. 129(8) 819-025 (2004)). Applicants have demonstrated that foe ms3 marker described by Park et ai. can be used for high-throughput analysis of DMA samples extracted fmm melon seeds (See Figure 10), Figure 10 provides an 20 illustration of an agarose gel showing resolution of alleles at foe marker linked to the ms3 gene. The ms3~steriie associated marker allele is foe upper band in lanes 6, 8,10, 12, 14,16-21 and foe ms3-fertiie associated marker allele is foe Sower band in lanes 1-5,7-9,11,13,15,17-21.

Applicants have also mapped the ms3 gene onto a genetic linkage map 25 (See Figure 11), Figure 11 provides an illustration of genetic linkage groups containing the ms3 arid ms4 genes showing positions of the ms genes relative to linked molecular markers- Linkage distances (in cM) are shown to: the left of 2014200607 05 Feb 2014 -47*·· linkage groups and moiecuiar marker-and ms iocs are shown to the right of linkage groups.

Applicants have also mapped the ms4 gene to a position between two microsateliite markers CmNR85 and CmG2797 (See Figure: 12). Figure 12 5 provides an illustration of an Agarose gel image showing PCR products from reaction mixes using DNA isolatedfrom melon seed tissue samples. Experience to date suggests that the markers Jinked to the ms3 and ms4 genes provide the necessary marker toots for efficient introgression of ms genes into female parent lines of melon as welt as for selection, using the methods of automated, 10 nondestmetive deed sampling and marker analysis described herein, of male sterile^ gehot^pes <ms/ms) ftom stock seed lots of female parent lines segregating for steriiity.

Advantages of using the methods of this disclosure include, without limitation, reduction of labor and field resources: required per population or 15 breeding tine, increased capacity to evaluate a larger number of breeding populations per field unit, and increased capacity to analyze breeding populations for desired traits prior to planting, Field resources per population are reduced by limiting the field space required to advance the desired genotypes. For example, a population of 1,000 individuals may be planted at 25 seeds per 20 row consuming a total Of 40 rows in the field. Using conventionaf tissue sampling, all 1,000 plants would be tagged and manually sampled by scoring leaf tissue, Molecular marker results would be needed prior to pollination and only those plants containing the desired genetic composition would be pollinated; Thus, if it was determined that S0 seeds contained the; desired genetic 25 composition, conventional breeding methodology would hive required the planting of 1000 plarits to retain the desired SO seeds. By contrast, the methods of this disclosure allow the breeder to analyze the 1,000 seeds in the lab-.and" 2014200607 05 Feb 2014 -48- select the 50 desired seeds prior to planting. The 50 individuals can then be planted only two 2$ seed rows. Additionally, the methods of this disclosure do not require tagging or sampling in tie field, thereby significantly reducing the required manual labor resources. 5 In addition to reducing the number of field rows per population, the methods ofthis disclosure may further increase tie number of populations the breeder can evaluate in a given breeding nursery. Using tie above example wherein 50 seeds out of each population of 1000 seeds contained hie desired genetic composition, a breeder applying the methods of this disclosure could 10 evaluate 20 popuSafions of 5() seeds each using the same field area consumed by a single population using convenfe sampling techniques. Even if the populations are selected for a single allele, using a 1:2:1 expected segregation ratio for an: F? population, the breeder could evaluate 4 populations in the same field area: as a single field tissue sampled population. IS A potential further advantage to the methods of the present disclosure is the mitigation of risks associated with growing plants in certain geographies where plants may grow poorly or experience poor environmental conditions including disease, or may even be destroyed during storms. * * # ;# * * .# * 20

When introducing elements or features of embodiments herein, the articles “a", “an", “the” and “said’' are intended to mean ihatthere are one or more of such elements or features. The terms -comprising", ‘'including" and “having" are intended to be inclusive and mean that there may be additional elements or 25 features other than those specifically noted, it is further to be understood that the method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order 2014200607 05 Feb

Ο <N discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may he employed.

The description of the diselosure is merely exemplary in nature and, thus;, variations that do notdepartfrom the gist of thedisclosureare intended to be within the scope of the disclosure. SuchX^ations are ned ip he regarded as adeparture from the spirit andseope of the disclosure. A reference herein to a patent document or other matter which is given as prior art is not taken as an admission that that document or prior art was part of common general knowledge at the priority date of any of the claims.

Claims (13)

1. The claims defining the invention are as follows:

   1. A method for testing individual seeds in a population for a trait of interest, the method comprising: providing a population of seeds; separating, via an automated seed singulator, individual seeds from the population of seeds; depositing and retaining each seed onto a corresponding one of a plurality of orientation pedestals; collecting orientation image data, via an orientation subsystem, of each seed retained on the respective orientation pedestal; orienting the individual seeds based on the collected orientation image data; removing a tissue sample comprising cells with nucleic acids from the individual separated and oriented seeds, via an automated sampling assembly, while preserving the germination viability of the seed; analyzing nucleic acids extracted from each tissue sample to determine the presence of at least one genetic marker indicating the presence of a genetic trait of interest; and selecting particular seeds from the individual sampled seeds based upon the determination of the presence of the at least one genetic marker in the particular seeds.
2. 2. The method of claim 1 wherein the seed comprises seed selected from the group consisting of com seed, soybean seed, wheat seed, cotton seed, cucumber seed, dry bean seed, garden bean seed, gourd seed, melon seed, pea seed, squash seed, sweet com seed, okra seed and watermelon seed.
3. 3. The method of claim 1 wherein the trait of interest comprises a male-sterile gene and wherein the method further comprises cultivating fertile plants from the selected seeds to introgress male sterility into seeds provided by the cultivated fertile plants.
4. 4. The method of claim 3, wherein the method further comprises utilizing the fertile plant as a female parent in a cross with another plant.
5. 5. An automated method for testing seeds for a trait of interest, the method comprising: automatically separating individual seeds from a plurality of seeds at a seed loading station of an automated seed processing system; automatically receiving the separated individual seeds at an orientation subsystem of the automated seed processing system; retaining each seed on one of a plurality of orientation pedestals of the orientation subsystem; collecting orientation image data of each seed retained on the respective orientation pedestal, via at least one imaging device of the orientation subsystem; automatically orienting each individual seed retained on the respective orientation pedestal utilizing the collected orientation image data; automatically removing a tissue sample comprising cells with nucleic acids from each of the oriented individual seeds, while preserving the germination viability of each seed, via an automated sampling subsystem of the automated seed processing system; automatically depositing each sampled seed into a selected well in a selected one of a plurality of seed collection trays after the seed has had the tissue sample removed, and depositing each tissue sample into a selected well in a selected one of a plurality of sample collection trays after the sample has been removed from the seed; storing in a database information identifying the selected well and the selected sample collection tray into which each tissue sample was deposited and the selected well and the selected seed collection tray into which each corresponding sampled seed was deposited, such that each sample and corresponding seed from which the sample was removed can be tracked to provide single seed identity of each seed to the corresponding sample removed therefrom; analyzing nucleic acids extracted from each tissue sample to identify the tissue samples having at least one genetic marker indicating the presence of a genetic trait of interest; and selecting particular seeds from the seed collection tray that correspond with the tissue samples identified as having the trait of interest utilizing the information stored in the database.
6. 6. The method of claim 5 wherein the seeds comprise seeds selected from the group consisting of corn seed, soybean seed, wheat seed, cotton seed, cucumber seed, dry bean seed, garden bean seed, gourd seed, melon seed, pea seed, squash seed, sweet corn seed, okra seed and watermelon seed.
7. 7. The method of Claim 5, wherein retaining each seed on an orientation pedestal comprises depositing each seed onto the corresponding one of the plurality of orientation pedestals of the orientation subsystem and retaining each seed on the respective orientation pedestal without damaging the embryo of each seed.
8. 8. The method of Claim 7, wherein automatically orienting each individual seed comprises collecting the orientation image data of each seed retained on the respective orientation pedestal and utilizing the collected orientation image data to orient each seed in a particular orientation without damaging the embryo of each seed.
9. 9. The method of Claim 8, wherein removing a tissue sample from the individual seeds comprises: depositing each oriented seed into a corresponding one of a plurality of sampling retention fixtures having a ‘Tip’ of each seed, containing the embryo, positioned away from a cutting device of each of a corresponding one of the plurality of sampling assemblies associated with the sampling retention fixtures, each sampling assembly associated with a corresponding one of the sampling retention fixtures; and firmly and securely holding each seed within the respective sampling retention fixture, without damaging the embryo of each seed, as each respective cutting device removes the tissue sample from a portion of each seed not containing the embryo.
10. 10. The method of Claim 9, wherein removing a tissue sample from the individual seeds further comprises abutting a ‘Crown’ of each seed against a corresponding one of a plurality of datum blocks associated with the sampling retention fixtures, each datum block associated with a corresponding one of the sampling retention fixtures, such that a particular amount of the ‘Crown’ of each seed is removed as the tissue sample without damaging the embryo of each respective seed.
11. 11. The method of Claim 5, wherein removing a tissue sample from the individual seeds further comprises: removing a first portion of each seed comprising the skin of each respective seed; and removing a second portion of each seed comprising tissue usable as the sample and suitable for the genetic analysis.
12. 12. The method of claim 5 wherein the trait of interest comprises a male-sterile gene and wherein the method further comprises cultivating fertile plants from the selected seeds to introgress male sterility into seeds provided by the cultivated fertile plants.
13. 13. The method of claim 12, wherein the method further comprises utilizing the fertile plant as a female parent in a cross with another plant.