WO2011163326A2

WO2011163326A2 - Automated seed chipping apparatus

Info

Publication number: WO2011163326A2
Application number: PCT/US2011/041388
Authority: WO
Inventors: Jason Abbas; John Woodford; Dean Ovel; Daniel Harms; James Jones
Original assignee: Syngenta Participation Ag
Priority date: 2010-06-22
Filing date: 2011-06-22
Publication date: 2011-12-29
Also published as: WO2011163326A3; WO2011163362A1

Abstract

An automated apparatus for removing a portion or chip of seeds in a research or commercial program for analysis is disclosed. Seed subject to analysis are placed in the apparatus. A vibrating table moves individual seeds to a pick-up station. Properly presented seeds are moved to a sampling station by an arm. A laser cuts off a portion or chip of the seed, leaving the seed viable. The removed chip drops into a preselected cell of a segmented tray and the seed is dropped into a different preselected cell of a segmented tray. The seed and chip are subjected to analysis for one or more selected characteristics. The results of the analysis are recorded, such as in a database, with the chip results or data correlated to the seed results or data so that research or commercial decisions regarding the seed are facilitated. The apparatus automates the chipping procedure greatly increasing the throughput of the sampling and greatly reducing the labor required to carry out the sampling.

Description

AUTOMATED SEED CHIPPING APPARATUS

This application claims priority to United States Patent Application Serial No. 61/426298 filed 22 December 2010 (22.12.2010) and United States Patent Application Serial No 61/357322 filed on 22 June 2010 (22.06.2010) each filed in the United States of America.

Background of the Invention

The present invention relates generally to apparatus for automating the analysis of seed and, more specifically, to apparatus for automating the removal of a portion of seed for subsequent analysis of the seed and the removed portion or chip.

Commercial seed companies are engaged in a continuous process to develop and sell seed that will grow into plants having improved agronomic and/or commercial properties. That continuous process includes the development of a very large number of novel plants that are evaluated for desired characteristics and selected for advancement if those characteristics meet the criteria of the seed company.

One technique that has developed to assist in the evaluation is the removal of a portion of a seed to be analyzed for the desired characteristics so that the portion can be analyzed by any desired means, including destructive means, while leaving the seed viable so that a plant can be grown from the seed and its progeny maintained in the research program. In the past, this process was done by hand and was very time consuming and labor intensive. Efficiencies in producing the chips and viable chipped seeds can be achieved by automating the process.

Summary of the Invention

The present invention consists of an automated apparatus for chipping seeds, producing both chipped seeds and the removed chip that are identified or correlated to each other for subsequent analysis and data processing. A collection of seeds to be sampled is loaded into the apparatus and the seeds are fed into a vibratory bowl. The vibratory isolation process of the vibratory bowl acts to move the individual seeds into a consistent and desired orientation while advancing the individual seeds toward a pick-up station. Seeds that are not properly oriented or spaced are detected and sent back to the vibratory bowl for later selection and presentation for chipping.

A properly oriented seed at the pick-up station is grasped by an arm that is controlled to be moveable between the pick-up station and a sampling or chipping station. Once positioned at the chipping station, a laser is controlled to sever a desired portion of the seed, oftentimes referred to as a chip, and the chip falls into a selected isolation cell where it is maintained separate from other chips. The arm then moves the chipped seed and drops it into a separate, selected isolation cell where it is maintained separate from other chipped seeds. The apparatus generates data that correlates the cells of the chip and the chipped seed. The cells may be cells of a segmented tray and wherein the apparatus is operable to select and use a tray from a cassette of stacked trays thereby improving the volume of seeds that can be automatically processed by the machine without human involvement.

An aspect of the present invention is to automate the chipping of seed, thereby reducing the time, space and labor required over manual seed chipping operations.

Another aspect of the present invention is the facilitation of analysis on the chip and possibly the chipped seed and concomitant generation of data that assists in making research decisions regarding use of the seed in a research program.

A further aspect of the present invention is the preservation of identity or correlation between the chip and the seed from which the chip was taken so that the results of the analysis on the chip can be identified or correlated to the seed.

Yet another aspect of the present invention is the reduction in time from selection of the seed for chipping to the generation of data for decision making thereby reducing the time required for making decisions.

Yet a further aspect of the present invention is the increase in throughput of seeds to allow the generation of an increased amount of data for the decision making process.

A method of the present invention comprises using a high-throughput, non-destructive method for analyzing individual seeds chips associated with a population of seeds which removes a seed chip from at least a subset of seeds in a seed population using the apparatus which cauterizes the seed chip and the sampled seed and it is preserving the cauterized seed germination viability of the one or more sampled seeds. The method has a step of analyzing the one or more seed chips and using the analysis data for selection of cauterized viable sampled seeds for further use.

Yet a further aspect of the method is that the one or more seed chip are analyzed for one or more characteristics indicative of at least one chemical or genetic trait.

Yet a still additional aspect of the method is that the method further comprises selecting one or more individual sampled seeds from the population of seeds based on the presence of one or more characteristics that are genetically linked with a haplotype associated with a qualitative trait locus (QTL).

Additionally, the method further comprises selecting one or more individual sampled cauterized seeds from the population of seeds based on the presence of one or more

characteristics that indicate association with a recurrent parent.

Yet further the method uses characteristics which are associated with presence or absence of one or more transgenes or transgenic events in the selection process.

And still further the method permits analyzing the one or more seed chip samples for one or more alleles and determining the ploidy level of the sampled cauterized viable seed.

Still another aspect of the present invention is the use of a vibratory bowl for the orientation, spacing and advancement of individual seeds to a pick-up station.

Still a further aspect of the present invention is the use of a translatable arm for moving individual seeds from the pick-up station to a chipping or sampling station.

Another aspect of the present invention is the use of a laser to reliably slice a selected portion off of the seed while simultaneously cauterizing the seed in the area of the removed portion to increase the likelihood of the chipped seed remaining viable for subsequent growth into a plant.

Another aspect of the present invention is the use of a tool for removing a portion of the seed which is moveable in the x,y,z directions for removing different portion sizes from different seeds. Brief Description of the Drawings

Fig. 1 is front, left-side perspective view of a preferred embodiment of the present invention, including the vibratory bowl and a vibratory rail for orienting seed.

Fig. 2 is a front, right-side perspective view of the preferred embodiment of Fig. 1, including the laser.

Fig. 3 is a perspective view of the preferred embodiment of Fig. 1 with access doors open to the interior.

Fig. 4 is an enlarged perspective view of the vibratory bowl and showing

soybean seeds advancing toward the pick-up station.

Fig. 5 is an enlarged, upper perspective view of the pick-up station and showing the arm for moving a selected seed from the pick-up station to the sampling station.

Fig. 6 is an enlarged perspective view of the sampling station.

Fig. 7 is an enlarged plan view of the sampling station.

Fig. 8 is rear, left-side perspective view of a tray handling system.

Detailed Description of the Preferred Embodiments

The invention will be described with respect to a particular embodiment and wherein soybean seeds are being sampled by the apparatus. A skilled artisan will recognize, however, that many other types of seeds can be sampled using the present apparatus, including but not limited to alfalfa, barley, canola, corn, cotton, cucumber, flax, flowers, melon, millet, oat, peanut, pepper, rice, rye, safflower, sorghum, sunflower, other vegetables and wheat.

In general, a collection of seeds for sampling by the apparatus is selected. The seeds are loaded into a vibratory bowl that advances the seeds toward a pick-up station. The vibratory action of the bowl acts to move the seeds into a consistent orientation so that each seed will be presented to the pick-up station generally in the same orientation to assure consistent sampling. In the preferred embodiment, a camera monitors each seed as it approaches the pick-up station and if the seed is not in the desired orientation, it is moved back into the vibratory bowl. When a properly oriented seed is present at the pick-up station, the apparatus moves an arm to the pickup station and a pair of fingers on the arm grasp the seed and move it to a sampling station. A laser severs a preselected portion of the seed and the removed portion is allowed to fall into a selected cell of a collection tray. While a laser is used to sever the chip in a preferred embodiment, other apparatus or techniques can be used, such as a blade, a knife, an air jet, or a water jet. The main portion of the seed is moved by the arm to a tube and dropped. The tube directs the main portion of the seed to a separate, selected cell of a collection tray. The apparatus outputs data that identifies or correlates the cells of the chipped or severed portion and the main portion of the seed for subsequent analysis. The chipped portions of the seeds are subjected to any variety of tests to assist in determining what role the individual seed will play in the research or commercial programs of the operator of the apparatus. Such tests may include cellular analysis, chemical analysis, hybridization analysis, nucleotide sequencing, and polymerase chain reaction (PCR) analysis. The main portion of the seed remains viable so that it can reproduce with the possibility that its progeny will constitute a part of the operator's research or commercial programs. EXAMPLE 1

Illustrated in Fig. 1, generally at 10, is an automated seed sampling apparatus representing a preferred embodiment of the present invention. The apparatus 10 includes a pair of access doors 12 and 14, a collection tray stacker 16, a computer control 18, supply and exhaust vents 20 and 22, and a vibratory bowl 24. The apparatus 10 also includes a laser system, indicated generally at 26 in Fig. 2.

The interior of the apparatus 10 is shown in Fig. 3 with the access doors 12 and 14 opened. The vibratory bowl 24 is located near the front of the apparatus 10. The vibratory bowl 24 has a spiral ramp 28 that leads from the interior of the bowl 24 outwardly and upwardly. The vibratory action of the bowl 24 causes soybean seeds 30 to climb the spiral ramp 28 and also serves to move the individual seeds into and common and consistent orientation. The spiral ramp 28 feeds the soybean seeds 30 onto a linear ramp portion 32 that terminates at a pick-up station 34 (Figs. 3 and 4). A camera 36 (Fig. 3) images each soybean seed 30 that approaches the pick-up station 34 and uses software to determine if the seed 30 is in the proper, predetermined orientation to be sampled. If the seed 30 is not properly oriented, a nozzle 38 (Fig. 4) shoots a jet of compressed air at the seed 30, knocking it off the linear ramp 32 and back into the vibratory bowl 24.

Seeds 30 with the proper orientation are allowed to progress up the linear ramp 32 to the pick-up station 34 where they are held stationary (Fig. 5). A sensor at the pick-up station 34 senses the presence of a seed 30 in position for sampling. Control apparatus activates an arm 40 that can move in the x,y,z directions and moves it to the location of the seed 30 at the pick-up station 34. When in the proper location, the control apparatus moves a pair of fingers 42 and 44 of the arm 40 to releasably grasp the seed 30. The seed 30 is then carried by the arm 40 to a sampling station 46. A beam from the laser 26 is directed in a predetermined pattern over the seed 30 severing a preselected portion of the seed 30 creating a removed portion. The removed portion falls into an opening 48 below the sampling station 46 down a tube to a collection location to be described in more detail below (Figs. 5 and 6).

After the portion of the seed 30 has been severed by the laser 26, the arm 40 is moved by the control apparatus to a location rearwardly of the sampling station 46 over an opening 50 and released. The seed 30 travels down a tube to a collection location to be described in more detail below.

Use of the laser 26 results in a certain amount of smoke and fumes being generated inside the apparatus 10. It is desirable to move air through the apparatus 10 using the supply and exhaust vents 20 and 22 to prevent an excess accumulation of smoke.

A collection tray indexing apparatus 52 is located generally below the sampling station

46 (Fig. 8). Although not shown in Fig. 8 for clarity purposes, each of a pair of collection trays having a two-dimensional array of cells are normally positioned on a corresponding one of a pair of wheeled trucks 54 and 56 and each are below a corresponding one of the covers 58 and 60. Truck 54 carries a collection tray for collecting and isolating in individual cells each removed portion of the seed as it is removed from the seed at the sampling station 46 by the laser 26. The apparatus 52 is translatable in an x direction and trucks 54, 56 are translatable in a y direction so that the collection trays can be moved to any desired position in the two-dimensional array of cells of the collection trays. Tube 62 communicates with the opening 48 in the seed sampling station 46 and directs the seed portion or chip into a selected cell of the collection tray on truck 54 that has been selected and positioned by the control apparatus using the apparatus 52 and the truck 54 to move the selected cell directly below the output of the tube 62. Similarly, tube 64 communicates with the opening 50 in the seed sampling station 46 and directs the chipped seed into a selected cell of the collection tray on truck 56 that has been selected and positioned by the control apparatus using the apparatus 52 and the truck 56 to move the selected cell directly below the output of the tube 64. The apparatus 10 maintains a record of the cell position and time of sampling for each chipped seed sampled and each corresponding seed chip. In this way, each sampled seed is uniquely identified and correlated to the corresponding seed chip so that information gathered from analysis on either the chipped seed or the seed chip or both can be tracked. By maintaining each chipped seed and corresponding seed chip in its corresponding column and row position in the collection trays, the identity of each chipped seed and corresponding seed chip can be maintained. This allows viable chipped seed identified to be of interest by virtue of the selected analysis method to be known and utilized in the relevant research or commercial program(s).

In the preferred embodiment, the collection tray stacker 16 sits adjacent the collection tray indexing apparatus 52 and serves as a holding station for both the unfilled and the filled collection trays. The stacker 16 is controlled to cycle unfilled collection trays to the collection tray indexing apparatus 52 and receive and store filled trays from the collection tray indexing apparatus 52. While use of the stacker 16 is not required, it greatly increases the quantity of seed that can be sampled automatically by the apparatus 10 without human oversight or intervention.

A laser suitable for use in the present invention is Model No. 48-2KAL, air-cooled sealed carbon dioxide (C0₂) 25 watt laser commercially sold by Synrad of Mukiteo, WA. The laser has typical characteristics and adjustability (e.g., power or intensity), other types of gas lasers could be used, as can other types of lasers. An optics package to focus and control the laser beam can be included. Typical power supply, control circuit, optics and accessories are available from

Synrad. The power and pattern of the beam necessary to remove the desired portion of the seed without materially reducing the viability of the chipped seed can be readily achieved by a skilled artisan. While the portion to be removed may vary from species to species or from assay to assay, typically the amount removed is enough to expose the targeted internal tissue of the seed without destroying it or materially damaging it. With respect to soybean seed, when testing the chips using single nucleotide polymorphisms (SNP) (endpoint or real-time analysis), it has been found suitable to remove the seed coat portion of the seed to expose the cotyledon. The skilled user needs to adjust several parameters on the laser, such as power, cutting speed, path length and position of cut relative to the seed edge to remove the necessary amount to perform analysis, but doing minimal harm to the seed. Inert gas (i.e. argon) is allowed to flow over the cutting site while the laser is energized to help minimize the charring of the remaining seed and the removed portion.

Because the seed chipping apparatus forms small samples of seeds while retaining a viable cauterized seed portion for planting use, the seed chip that can be utilized in methods to improve germplasm without loss of the seed's genetics in the breeding program. This device is adapted to chip any number of different seeds with different seed shapes, weights and sizes. Seeds of fruits such as apple, pear, oranges; vegetable seed such as cucumber, tomato, pea, bean, corn, carrot and the like can be sampled. Oil seeds, fiber seeds, crop seeds like maize, soybean, rice, canola, sunflower, sugar beet, rice forage and grass seeds, tree seeds, flower seeds, weed seeds, ornamental seeds, thyme and other spice seeds can be sampled with this invention and method. The seed samples from any seed are useful in these improvement methods. Analyzing the samples identifies traits, or genotypes permitting the desireable viable seeds to be identified and selected. The analytical methods test chemicals or genetics of the samples from batch or bulk population of seed samples.

The sample most often contains endosperm tissue that allows detection of alleles that are useful in detection of the parental source for a marker or specific. Testing alleles and their frequencies in samples from different germplasm types can be employed to build ancestor or genetic linkage maps. Allele frequencies also provide the ability to detect linked and unlinked traits.

The use of the laser in this seed sampling acts to seal the viable seeds wound. This is particularly useful in seeds that are not fully dented and dried. The seed sample can be mixture of seed types where at least some are putative haploid seed. This sample can be tested to identify the ploidy characteristic of the sample. Additionally the haploid or diploid samples can be analyzed for genotypic information, traits and chemicals. The correlated seed for the sample can be used in germplasm improvement through selection of seeds for breeding or markers assisted breeding. The haploid seeds can be doubled, forming doubled haploids and the resultant doubled haploid seeds can also be chipped and the sample can be analyzed selected based on

phenotypical, morphological, genotypical characteristics detectible with markers.

This automated seed sampling device is part of a program to select associated plants or seeds with a haplotype, sequence, DNA, R A, transcription profile, and methylation pattern. The seed progeny of the selected seeds can also be automatically sampled in a repeated cycle that produces breeding and development economies of scale. Breeding methods are selected based on the type of sampled material hybrid, inbred, elite, non-elite, the type of characteristic desired, its heretability, its genetic complexity, etc. The sample can be analyzed for markers associated with most of the breeding traits like, water useage, vigor, emergence, yield, moisture, stress and disease tolerance, flowering, seed, pollination or harvest traits, and other characteristics.

This is often referred to as marker-assisted breeding program using a population of the analyzed chips and selected seeds with the trait, marker associated with the trait or a selected genotype. This program will use less land and personal resources while permitting seed bulking to occur sooner in the process.

In one embodiment, the automated seed chipping method produces a chip for analyzing a population of seeds by extracting a sample of nucleic acids from the populations of chips testing the for at least one genetic marker; forming a new selected seed population using the analysis; and producing plants from the selected seed. The present invention has a seed rail that orients the seed for chipping, because the seeds are positioned correctly less germination viability is lost in the chipping process. While seed germ ability prior to being chipped is the most important factor in seed viability, laser sealing may increase this germination viability. However, given that the seed has good germ, the automated orientation results in large quantities of viable seed portions. It is highly desirable to have not less than 50% remain viable after sampling. Viability seed portion rates, after being lasered, of 70%, 75%, and 80% and higher are most desired. In some circumstances, low viability rates are still economically useful. In breeding programs it is important to maintain germination viability planting season. Chemical treatments, biological treatments, polymers, fungicides, and pesticides known in the art for protecting a seed from environmental conditions while in storage or transport can be employed to assist in maintaining viability shelf life.

Samples prepared by the present invention can be used for testing chemicals such as proteins, peptides, oils, oil and fatty acid profiles, carbohydrates, amino acids, starches, and starch profiles, metabolites, storage proteins, introduced traits (whether GMO or mutated), and the like. The genetic characteristics of the seed sample can also be investigated. This investigation can employ detection means that are indicative of genetic traits like markers, single nucleotide polymorphisms, SSR, haplotypes, DNA-derived sequences, RNA-derived sequences, promoters, terminators, introns, untranslated regions of genes, microRNA, siRNA, transgenes, genes, mRNA, ds mRNA, transcriptional profiles, and methylation patterns. DNA may be extracted from the sample through use of SDS and a centrifugation and then amplified after extraction using methods like DNA amplification prep from Amersham Biosciences. RNA may be extracted from the sample using methods known to those of skill in the art.

Nucleic acids extracted from the sample of seed are tested for polymorphism using a wide variety of technologies known to those skilled in the art. Detection of the presences or the absences of insertions or deletions in sequence, simple repeats of sequences, single nucleotide or feature polymorphisms, whole or partial sequences or transcription profiles are used to select seeds for research or breeding. The selected seeds can be grown and their seedlings can likewise tested with leaf punch assays for genes, QTL, alleles, or genomic regions (haplotypes) that comprise or are linked to a genetic marker. This is particularly useful if one or more of the traits or characteristics are more readily detected in the nonseed tissues. Standard analysis of the material whether seed or leaf include Taq Man assays for PCR detections, sequencing methods, chips and microarray methods can be used to generate data for the selection and breeding decisions. The seeds can be tested selected and used in a breeding program. This can include repeated seed sampling tests with or without leaf assay testing for further selection and efficiencies. Seeds often are selected because of characteristics that are genetically linked with a QTL such as stress tolerance, water, nutrient optimizing characteristics, resistances to pesticides, diseases and insects, viruses, fungi or nematodes, altered fatty acid profiles, selected sugar or starch or other carbohydrate metabolisms, yield traits, increased/decreased oil, daylength, growth, germination, and other agronomic, commercially useful or commodity traits. The selected seeds are planted and bred with or selfed for development of progeny seed, which can be sampled, analyzed and a set selected for further processes to develop germplasm, variety, cultivar, inbred, hybrid, 3-way cross or haploids or the seeds of any of these. The breeding cycle can employ any number of breeding methods and breeding technologies in association with the sampling. A seed can be sampled once in the breeding process or numerous generations can be sampled and selected depending on the need for selection information.

Upon completion of the one or more selected methods of analysis, the results can be used to select one seed over another, or select a seed because of its trait. Accordingly, removal of a portion of a seed without destroying the viability of the seed allows selections to be made quickly and with a great savings in labor and resources in that the seed does not first have to be planted so that plant tissue can be analyzed.

The foregoing description and drawings comprise illustrative embodiments of the present inventions. The foregoing embodiments and the methods described herein may vary based on the ability, experience, and preference of those skilled in the art. Merely listing the steps of the method in a certain order does not constitute any limitation on the order of the steps of the method. The foregoing description and drawings merely explain and illustrate the invention, and the invention is not limited thereto, except insofar as the claims are so limited. Those skilled in the art that have the disclosure before them will be able to make modifications and variations therein without departing from the scope of the invention.

Claims

We claim:

1. Apparatus for the automation of sampling of seeds, comprising:

(a) a first target seed position

(b) a graspable member to grasp a seed at the first target location and move it to a second target location;

(c) a tool for removing a portion of the seed producing a chip and a chipped seed;

(d) a selectable collection receptacle for the chip; and

(e) a selectable collection receptacle for the chipped seed.

2. The apparatus of claim 1, wherein the tool is selected from the group consisting of a blade, a knife, an air jet, a water jet and a laser.

3. The apparatus of claim 1, wherein the collection receptacle is a segmented tray.

4. The apparatus of claim 1, further comprising a memory device for recording the collection receptacle of the chip and the chipped seed.

5. The apparatus of claim 1, wherein the selectable collection receptacle of the chipped seed is separate from the selectable collection receptacle of the chip.

6. A high-throughput, non-destructive method for analyzing individual seeds chips associated with a population of seeds, the method comprising the steps of: removing a seed chip from at least a subset of seeds in the seed population using an automated sampler which cauterizes the seed chip and the sampled seed while preserving the germination viability of the one or more sampled seeds; and analyzing the one or more seed chips and using the analysis data for selection of sampled seeds for further used.

7. The method according to claim 6, wherein the one or more seed chips are analyzed for one or more characteristics indicative of at least one chemical or genetic trait.

8. The method according to claim 7, wherein the method further comprises selecting one or more individual sampled seeds from the population of seeds based on the presence of one or more characteristics that are genetically linked with a haplotype associated with a QTL.

9. The method according to claim 7, wherein the population has a recurrent parent and the method further comprises selecting one or more individual sampled seeds from the population of seeds based on the presence of one or more characteristics that indicate association with the recurrent parent.

10. The method according to claim 7, wherein characteristics is associated with presence or absence of one or more transgenes or transgenic events.

11. The method according to claim 7, wherein the method further comprises: analyzing the one or more seed chip samples for one or more alleles; and determining the ploidy level of the sampled cauterized viable seed.

12. The apparatus for the automation of sampling of seeds according to claim 1, wherein the tool for removing a portion of the seed producing a chip and a chipped seed produces a cauterized viable chipped seed.

13. The apparatus for the automation of sampling of seeds according to claim 1, wherein the tool for removing a portion of the seed is moveable in the x, y, z directions for removing different portion sizes from different seeds.