Patent Abstract:
A method of accumulating a quantity of seeds having a desired fatty acid characteristic is provided. The method includes removing a sample from each seed in a population of seeds while maintaining the germination viability of the seeds; contacting each sample with a solvent to form a mixture comprising fatty acid methyl esters; analyzing the mixture of fatty acid methyl esters from each sample to determine the fatty acid profile of the corresponding seed; selecting seeds having at least one desired fatty acid characteristic based on the analysis of the samples removed from the seeds; cultivating plants from the selected seeds; recovering seeds from the cultivated plants, wherein the recovered seeds are a subsequent generation of the selected seeds; and repeating the operations for one or more generations of the recovered seeds to thereby accumulate the quantity of seeds having the desired fatty acid characteristic.

Full Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 11/510,771, filed Aug. 25, 2006,which claims priority to U.S. Provisional Application Ser. No. 60/711,775, filed Aug. 26, 2005.The entire disclosures of each of these applications are incorporated herein by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     This invention relates to systems and methods for the high throughput screening and identification of fatty acid composition signatures in biological materials such as seeds. 
     Oil seeds are valuable crops with many nutritional and industrial uses due to their unique chemical composition. Accordingly, seed breeders are continually trying to develop varieties of oil seeds to maximize oil seed yield and/or production. As such, grain handlers and seed breeders must be able to distinguish an oil seed from a regular seed to make important decisions in a grain handling situation or in a seed breeding operation. Such decisions have traditionally been based on statistical sampling of a population of seeds because determining the fatty acid characteristics of a population of seeds has been laborious and time consuming. However, statistical sampling necessarily allows some seeds without the desirable trait to remain in the population, and also can inadvertently exclude some seeds from the desired population. 
     Thus, there is a need for high throughput screening systems and methods for use in the identity testing of oil seeds. 
     SUMMARY OF THE INVENTION 
     The present invention relates to systems and methods for screening seeds to determine their fatty acid characteristics. The systems and methods are particularly adapted for high-throughput and automation, which permits greater sampling than was previously practical. Further, the high-throughput, automated and non-destructive sampling permitted by at least some of the embodiments of this invention allow for the screening and testing of every seed in a population, whereby the seeds that do not express the desired fatty acid characteristics can be culled. Further, embodiments of this invention are fully transportable such that testing of most or all of the seeds in a population can be completed in the field. Thus, the rapid assays provided by the present invention, which typically require less than about 10 minutes total analysis time, are ideally suited for the identity testing of oil seeds at grain elevators, oil processing plants, food formulations laboratories and the like or in seed breeding applications where large numbers of small samples must be analyzed to make immediate planting decisions. Accordingly, the systems and methods of the present invention greatly speed up the process of evaluating a population of seeds, for example, in making effective purchasing or handling decisions in the field or in making planting decisions when bulking a given seed population in a breeding program so that time and resources are not wasted in growing plants without desired traits. 
     Generally a method of this invention for determining the fatty acid composition of a plurality of seeds comprises sequentially feeding a seed to a sampling station; holding the seed in a sampling station; scraping a sample from the seed being held in the sampling station; conveying the sample to an individual compartment in a sample tray; extracting oil from the sample in the sample tray; transesterifying extracted oil from the sample in the sample tray to form a mixture of fatty acid esters; and analyzing the mixture of fatty acid esters from the sample to determine the fatty acid profile of the corresponding seed. 
     The invention is also directed to a method for high throughput screening of oil seeds. The method comprises providing tissue samples from a plurality of oil seeds in individual compartments of a sample tray; contacting each tissue sample in the sample tray with toluene to produce a mixture comprising fatty acid methyl esters; analyzing the mixture of fatty acid methyl esters from each sample to determine the fatty acid profile of the corresponding oil seeds; and selecting seed based on the presence or absence of a desired fatty acid characteristic. 
     The invention further provides a system for high throughput screening of fatty acid composition in a seed. The system comprises a sampling station for holding an individual seed; a sampling mechanism for removing material from a seed in the sampling station; a seed feeder for feeding individual seeds to the sampling station; a sample transport for transporting the sample from the sampling station to a fixed location; a table for supporting at least one sample tray having a plurality of compartments for holding individual samples from individual seeds, the sample trays being further adapted to accept a volume of solvent suitable for extracting and converting oil in the samples to a mixture of fatty acid esters; and means for analyzing the mixture of fatty acid esters for each sample to determine the fatty acid profile of the corresponding seeds. 
     The invention further provides a method of bulking up a quantity of seed having a desired fatty acid characteristic. The method comprises (a) removing a sample from each seed in a population without affecting the germination viability of the seeds; (b) contacting each sample with a solvent to form a mixture comprising fatty acid methyl esters; (c) analyzing the mixture of fatty acid methyl esters from each sample to determine the fatty acid profile of the corresponding seed; (d) selecting seeds having at least one desired fatty acid characteristic;(e) cultivating plants from the selected seeds; (f) recovering seed from the cultivated plants; and repeating steps (a) through (f) for one or more generations. 
     These and other features and advantages will be in part apparent, and in part pointed out hereinafter. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a perspective view of one embodiment of an automated seed sampler system for use according to the principles of this invention; 
         FIG. 2  is an enlarged perspective view of the seed sampler assembly of the seed sampler system; 
         FIG. 3  is an enlarged perspective view of the hopper and seed feeding mechanism of the seed sampler assembly; 
         FIG. 4  is a perspective view of the broach for scraping samples from the seeds; 
         FIG. 5  is a perspective view of the slide for driving the broach of the piston actuator from the seed feeding mechanism; 
         FIG. 6  is a perspective view of the piston in the feed mechanism of the hopper; 
         FIG. 7  is a perspective view of the stage with a plurality of seed trays and sample trays mounted thereon; 
         FIG. 8  is a perspective view of the two-dimensional translation mechanism; 
         FIG. 9  is a perspective view of the inlet of the seed conveyor; 
         FIG. 10  is a perspective view of the outlet of the seed conveyor; 
         FIG. 11  is a perspective view of the outlet of the sample conveyor; 
         FIG. 12  is a perspective view of the air multiplier used in the seed and sample conveyors; 
         FIG. 13  is a top plan view of a high throughput seed sampler system for use in accordance with the principles of this invention; 
         FIG. 14  is a side elevation view of the high throughput seed sampler device; 
         FIG. 15  is a front perspective view of the seed sampler system; 
         FIG. 16  is a rear perspective view of the seed sampler system; 
         FIG. 17  is a perspective view of the sampling station of the high throughput seed sampler device; 
         FIG. 18A  is a partial perspective view of one portion of the seed sampling station in accordance with the principles of this invention, with the broach retracted; 
         FIG. 18B  is a partial perspective view of one portion of the seed sampling station in accordance with the principles of this invention, with the broach extended; 
         FIG. 19A  is a side elevation view of the seed sampling station, with the broach in its retracted position; 
         FIG. 19B  is a side elevation view of the seed sampling station, with the broach in its extended position; 
         FIG. 20  is a longitudinal cross-sectional view of the seed sampling station; 
         FIG. 21  is a front end elevation view of the seed sampling station; 
         FIG. 22  is a transverse cross-sectional view of the seed sampling station; 
         FIG. 23A  is a side elevation view of the seed selecting wheel; 
         FIG. 23B  is an exploded view of the seed selecting wheel; 
         FIG. 23C  is a vertical cross sectional view of the seed selecting wheel; 
         FIG. 24  is a front elevation view of the feeding mechanism; 
         FIG. 25  is a side elevation view of the feeding mechanism; 
         FIG. 26A  is a perspective view of the feeding mechanism; 
         FIG. 26B  is a side elevation view of the feeding mechanism; 
         FIG. 26C  is a longitudinal cross-sectional view of the feeding mechanism, taken along the plane of line  26 C- 26 C; 
         FIG. 26D  is a bottom plan view of the feeding mechanism; 
         FIG. 27A  is a vertical longitudinal cross-sectional view of the sampling mechanism; 
         FIG. 27B  is an enlarged partial vertical cross sectional view of the sampling mechanism as shown in  FIG. 27A ; 
         FIG. 28A  is a vertical transverse cross-sectional view of the sampling mechanism; 
         FIG. 28B  is an enlarged partial cross-sectional view of the sampling mechanism as shown in  FIG. 28A ; 
         FIG. 29  is a chromatogram of fatty acid esters obtained from a normal soybean in accordance with the method described in Example 1; and 
         FIG. 30  is a chromatogram of fatty acid esters obtained from a low linolenic acid soybean in accordance with the method described in Example 1. 
     
    
    
     Corresponding reference numerals indicate corresponding parts throughout the several views of the drawings. 
     DETAILED DESCRIPTION 
     The present invention provides methods for screening populations of biological materials such as seeds to determine their fatty acid characteristics. In an aspect of the invention, the analytical methods allow individual seeds to be analyzed that are present in a batch or a bulk population of seeds such that the fatty acid characteristics of the individual seeds can be determined. 
     In an embodiment of the invention for screening seeds, the methods of the present invention generally comprise extracting oil from a seed tissue sample and transesterifying the extracted oils to produce a mixture of fatty acid esters from each sample. The mixture of fatty acid esters is then analyzed by separating and detecting the fatty acid esters to determine a profile of fatty acid characteristics for each sample. These profiles can then be correlated to fatty acid profiles prepared from seeds of known origin in order to determine the fatty acid characteristics of the sampled seed. In a preferred embodiment, less than about 10 mg of seed tissue, and particularly less than about 5 mg of seed tissue, is sampled from the seed to maintain seed viability as further described below. 
     The extraction of oils from the sample can be conducted using any suitable solvent known in the art for extracting oil from a seed tissue. Preferably, the selected solvent is suitable for directly extracting and transesterifying oils to a mixture of fatty acid esters. Examples of suitable solvents for the direct extraction and transesterification of oils in the seed sample include without limitation, hexane, benzene, tetrahydrofuran, dimethyl sulfoxide, trimethylsulfonium hydroxide, petroleum ether, methylene chloride, and toluene. In a preferred embodiment, the solvent comprises toluene. 
     In a preferred embodiment, the method comprises simultaneously contacting a plurality of seed tissue samples with solvent in individual wells of a multi-well sample plate. For example, to increase throughput and sample handling, samples are preferably contacted with solvent in 96-well or 384-well microtiter plates adapted to accept a volume of solvent sufficient to wet the sample and complete the extraction and transesterification reactions. 
     The mixture of fatty acid esters produced from the extraction and transesterification reactions is then analyzed to determine the fatty acid characteristics of the individual samples. Such analysis may generally be conducted using any suitable means for separating and detecting the fatty acid esters present in the mixture. Preferably, such separation and detection is completed in less than about 5 minutes, more preferably less than about 3 minutes, so as to maintain throughput. In a particular embodiment, the analysis is conducted using a high speed gas chromatograph with flame ionization detection. An example of such an analysis system is gas chromatography using a Supelco Omegawax column (commercially available from Supelco, Inc., Bellefonte, Pa.). In a further preferred embodiment, the separation and detection is completed using direct headspace analysis to further increase throughput. 
     Thus, a particular embodiment for high throughput screening of a seed comprises providing tissue samples from a plurality of seeds in individual compartments of a sample tray; contacting each tissue sample in the sample tray with a solvent to produce a mixture comprising fatty acid esters; and analyzing the mixture of fatty acid esters from each sample to determine the fatty acid profile of the corresponding seeds. 
     In a preferred embodiment, the fatty acid profile of the corresponding oil seed is determined in less than about 10 minutes from the time in which an individual tissue sample is contacted with solvent. 
     The methods and systems of the present invention can be used to screen oil seeds such as soybean, corn, canola, rapeseed, sunflower, peanut, safflower, palm and cotton for a wide variety of fatty acid characteristics. For example, in one embodiment, a population of soybeans can be screened to determine the linolenic acid content, stearidonic acid (SDA) content, stearic acid content, oleic acid content, and saturated fat content of individual seeds. In another particular embodiment, a population of rapeseed can be screened to determine erucic acid content, oleic acid content, linolenic acid content, and the saturated fat content of individual seeds. Still further, in another particular embodiment, a population of sunflower can be screened to determine the oleic acid content, stearic acid content, and saturated fat content of individual seeds in the population. 
     In a particular embodiment, the methods of the present invention are used to determine the fatty acid characteristics of seeds in a breeding program. Such methods allow for improved breeding programs wherein nondestructive direct seed sampling can be conducted while maintaining the identity of individuals from the seed sampler to the field. As a result, the breeding program results in a “high-throughput” platform wherein a population of seeds having desired fatty acid characteristics can be more effectively bulked in a shorter period of time, with less field and labor resources required. Such advantages will be more fully described below. 
     As described above, particular embodiments of the sampling systems and methods of this invention protect germination viability of the seeds so as to be non-destructive. Germination viability means that a predominant number of sampled seeds, (i.e, greater than 50% of all sampled seeds) remain viable after sampling. In a particular embodiment, at least about 75% of sampled seeds or at least about 85% of sampled seeds remain viable. 
     In another embodiment, germination viability is maintained for at least about six months after sampling to ensure that the sampled seed will be viable until it reaches the field for planting. In a particular embodiment, the methods of the present invention further comprise treating the sampled seeds to maintain germination viability. Such treatment may generally include any means known in the art for protecting a seed from environmental conditions while in storage or transport. For example, in one embodiment, the sampled seeds may be treated with a polymer and/or a fungicide to protect the sampled seed while in storage or in transport to the field before planting. 
     The selected seeds may be bulked or kept separate depending on the breeding methodology and target. For example, when a breeder is screening an F 2  population for fatty acid characteristics, all individuals with the desired fatty acid profile may be bulked and planted in the breeding nursery. 
     Advantages of using the screening methods of this invention include, without limitation, reduction of labor and field resources required per population or breeding line, increased capacity to evaluate a larger number of breeding populations per field unit, and increased capacity to screen breeding populations for desired traits prior to planting. Field resources per population are reduced by limiting the field space required to advance the desired phenotypes. 
     In addition to reducing the number of field rows per population, the screening methods of this invention may further increase the number of populations the breeder can evaluate in a given breeding nursery. 
     The methods of the present invention further provide quality assurance (QA) and quality control by assuring that unwanted fatty acid composition characteristics are identified prior to a grain handler making purchasing or processing decisions or a seed breeder making planting decisions. 
     In a preferred embodiment, the methods of the present invention are used with an automated seed sampler system as described, for example, in U.S. Patent Application Publication No. US2006/0042527,filed Aug. 26, 2005,which is incorporated herein by reference. 
     An example of an automated seed sampler system suitable for use in the present invention is indicated generally as  20  in  FIG. 1 . The seed sampler system  20  is adapted to isolate a seed from a hopper, feed it to a sampling station, scrape a sample from the seed, convey the sample to a sample container, and convey the seed to a corresponding seed container. As shown in  FIG. 1 , the seed sampler system comprises a support  22 , a frame  24  on the support; a sampler assembly  26 , a stage  28  mounted on a two-dimensional translation mechanism  30 , a seed conveyor  32  for transporting seeds from the seed sampler assembly, and a sample conveyor  34  for transporting a sample removed from a seed to the seed sampler assembly. 
     As shown in  FIG. 1 , in the first preferred embodiment the support  22  comprises a wheeled cart  40 , having a four of vertical posts  42  connected by upper and lower longitudinal members  44  and  46 , at the front and back, and upper and lower transverse members  48  and  50  at the left and right sides, and a table top  52  mounted therein. A caster  54  can be mounted at the bottom of each post  42  to facilitate moving the support  22 . The details of the construction of the support  22  are not critical to the invention, and thus the support  22  could have some other configuration without departing from the principles of this invention. 
     As also shown in  FIG. 1 , the frame  24  comprises four vertically extending stanchions  60  mounted the table top  52 , which support a generally horizontal plate  62 . The sampler assembly  26  is mounted on the plate  62 , as described in more detail below. An arbor  64  is also mounted on the plate, and extends generally horizontally therefrom. The free end of the arbor  64  has first and second vertical posts  66  and  68  for mounting a seed conveyor  32  and parts of the sample conveyor  34 , respectively. The details of the construction of the frame  24  are not critical to the invention, and thus the frame could have some other configuration without departing from the principles of this invention. 
     As shown in  FIGS. 1 and 2 , the sampler assembly  26  is mounted on the plate  62  of the frame  24 . The sample assembly comprises a bin or hopper  70 , a sampling station  72 , and a feed mechanism  74  for delivering a single seed from the hopper  70  to the sampling station. 
     As shown in  FIGS. 1 and 3 , the stage  28  is adapted to securely mount a plurality of seed trays  80  and sample trays  82  in fixed positions and orientations. Each of the seed trays  80  and sample trays  82  is preferably divided into a plurality of compartments. The number and arrangement of the compartments in the seed trays  80  preferably corresponds to the number and arrangement of the compartments in the sample trays  82 . This facilitates the one-to-one correspondence between a seed and its sample. However, in some embodiments it may be desirable to provide multiple compartments in the sample tray for each compartment in the seed tray, for example where multiple tests may be run on the samples, or where different samples may be taken from the same seed (e.g. samples from different depths). 
     In a preferred embodiment, the sample trays  82  comprise multi-well microtiter plates. For example, the sample trays  82  may comprise a microtiter plate having a plurality of wells, preferably at least 96 wells and more preferably 384 wells per sample tray. Further, the wells of the microtiter plate are preferably adapted and/or sized to accept a volume of solvent suitable for extracting and converting oil in the samples to a mixture of fatty acid ethyl esters. 
     The stage  28  is mounted on a two-dimensional translation mechanism  30 , which in this preferred embodiment comprises a base  90  with a first linear actuator  92  having a translatable carriage  94  mounted on a base  90 , and a second linear actuator  96 , having carriage  98  mounted on the carriage  94  of the first linear actuator  92 . The stage  28  is mounted on carriage  98  of the second linear actuator  96 , and thus can be moved precisely in two dimensions through the operation of the first and second linear actuators  92  and  96 . 
     The seed conveyor  32  comprises a tube  100  with an inlet end  102  adjacent the sampling station  72 , and an outlet end  104  mounted on the post  66  of the frame  24 . There is a first venturi device  106  at the inlet end  102  of the tube  100  for inducing an air flow in the tube toward the outlet end  104  of the tube, and a second venturi device  108  at the outlet end  104  of the tube  100  for inducing an air flow toward the inlet end  102  of the tube. The first venturi device  106  is operated to create an air flow in the tube and draw a seed from the sampling station into the tube along the first end. The second venturi device  108  is then operated to create an air flow in the opposite direction, thereby slowing the seed down to reduce damage to the seed as it exits the outlet end  104  of the tube and is delivered to a compartment in the tray. In this preferred embodiment the second venturi  108  actually stops the movement of the seed, allowing it to drop under gravity to its compartment on a tray  90 . Various position sensors can be provided on the tube  100  to detect the presence of the seed, and confirm the proper operation of the seed conveyor  32 . 
     The sample conveyor  34  comprises a tube  120  with an inlet end  122  adjacent the sampling station  72 , and an outlet end  124  mounted on the post  68  of the frame  24 . There is a first venturi device  126  at the inlet end  122  of the tube  120  for inducing an air flow in the tube toward the outlet end  124  of the tube. A separator  128  is provided at the outlet end to separate the sample material from the air stream carrying it, so that the air stream does not blow the sample out of the compartment in the tray  92 . The separator preferably also contains a filter to prevent cross-contamination of the samples. 
     As shown in  FIG. 2 , the seed sampling assembly  26  is adapted to be mounted on the plate  62  on a post  140 . The seed sampling assembly  26  comprises a hopper mounting plate  142 , a slide mounting plate  144  and four slide standoff supports  146  therebetween. The hopper  70  (shown in  FIG. 3 ), which feeds individual seeds to a sampling station  72 , is mounted on the hopper plate  142 . The sampling station  72  comprises a seed nest  148  mounted on a nest mount  150 , which is supported from the slide mounting plate  144  by a pair of standoffs  152 . The nest  148  has a recess opening to its bottom surface, into which the hopper  70  feeds a single seed. There is a slot in the top of the seed nest  148  through which a portion of a seed in the recess is exposed. A broach  154  ( FIG. 4 ) is mounted in a broach holder  156  which is mounted on a slide transition plate  158  on a programmable slide  160 , with a broach clamping block  162 . The programmable slide  160  ( FIG. 5 ) is mounted on the underside of the slide mounting plate  144 , and moves the broach  154  through the slot in the seed nest  148  to remove a sample from a seed in the recess in the seed nest. 
     As best shown in  FIG. 4  the broach  154  as a plurality of teeth  164  that increase in height toward the proximal end, so that as the broach  154  is advanced in the slot, in cuts increasingly deeper into the seed in the recess in the nest  150 . The resulting gradual shaving reduces the damage to the seed, protecting its viability. Moreover, as described in more detail below, by cutting at different depths at different times, samples from different depths of the same seed can be separated for separate analysis. 
     A sample transfer tube  166  extends from the recess in the seed nest  148 , and has a connector  168  on its end for connection to the sample conveyor  34 . 
     The sampling station  26  also includes a hopper  70 , shown best in  FIG. 3 . The hopper  70  comprises left and right hopper mounting plates  170  and  172 , and a cylinder mounting plate  174  and a upper cylinder bracket  176 . The hopper  70  also has a front panel  178 , a back panel  180 , first and second end panels  182  and  184 , and bottom  186 . A divider  188  divides the hopper into first and second compartments  190  and  192 . The first compartment  190  holds a supply of seeds which are individually transferred to the second compartment  192 . 
     A piston actuator  194  operates a piston  196  to lift a seed out of the first compartment. A air jet assembly  198  transfers a seed from the end of the piston  196  to the second compartment  192 . The second compartment has a shaped bottom  200 , with a well  202  for receiving the seed and positioning it. A piston actuator  210  operates a piston  214  to lift a seed out of the second compartment  192 . An air jet assembly  216  is used to stir the seeds during the seed pick up procedure. 
     As shown in  FIG. 7 , the stage  28  has brackets  220  for mounting seed trays  90  and sample trays  92  in registration so that the seed conveyor and the sample conveyor deliver seeds and samples to corresponding compartments, in the respective trays. The sample trays  92  can (as shown) be adapted to hold individual vials. Of course, trays of different configurations could be used, for example where multiple compartments are provided for multiple samples from the same seed. For example where one sample is divided into several samples, or where the samples are separated from where they are taken, e.g. by depth. 
     As shown in FIG,  8 , the two-dimensional translation mechanism  30  also includes a slider  230  having a rail  232  and a carriage  234  that is positioned parallel to the first linear actuator  92 . The second linear actuator  96 , is mounted on the carriage  94  having carriage  98  mounted on the carriage  94  of the first linear actuator  92 . The stage  28  is mounted on carriage  98  of the second linear actuator  96 , and thus can be moved precisely in two dimensions through the operation of the first and second linear actuators  92  and  96 . Under appropriate control the translation mechanism can align individual compartment of the seed trays  90  and sample trays  92  with the outlets of the seed conveyor and sample conveyer. 
     As shown in  FIG. 9 , at the inlet end  102  of the tube  100  of seed conveyor  32 , a bracket  240  mounts an air amplifier  242  and a seed sensor tube  244 . The bracket  240  comprises sections  246 ,  248 ,  250 ,  252  and  254 . As shown in  FIG. 2 , the bracket  240  is mounted on the hopper mounting plate  142 . The air amplifier  242  (shown in  FIG. 12 ) is adapted to be connected to a source of compressed air, when air is applied to the air amplifier, it induces an air flow through the tube  100 , employing the venturi effect. The Sensor tube  244  and carries seed sensors  256  for sensing the passage of a seed therethrough. The sensors  256  are preferably optical sensors aligned with openings in the sensor tube  244  which optically detect the passage of a seed. 
     As shown in  FIG. 10 , a seed discharge assembly  260  is disposed at the outlet end  104  of the tube  100  of seed conveyor  32 . The discharge assembly is mounted on post  66 , with a bracket  262  and a discharge support  264 . A seed sensor tube  266  is mounted in the bracket  262 , and carries seed sensors  268  for sensing the passage of a seed therethrough. The sensors  268  are preferably optical sensors aligned with openings in the sensor tube  266  which optically detect the passage of a seed. An air amplifier  270  is connected to the seed sensor tube  266 . The air amplifier  270  ( FIG. 12 ) is adapted to be connected to a source of compressed air, when air is applied to the air amplifier, it induces an air flow through the tube  100 , employing the venturi effect. Below the air amplifier  270  is a connector tube  272 , and below that is a vented seed discharge tube  274 , which is also supported by a seed discharge tube holder  276 , carried on a seed discharge tube actuator  278 . 
     The inlet end  122  of the tube  120  of the sample conveyor  34  is connected via connector  168  to the sample discharge tube  166 . As shown in  FIG. 11 , the outlet end  124  of the tube  120  is connected to a sample amp connector  280 , which in turn is connected to air amplifier  282 , which is connected to chip nozzle assembly  284 . The chip nozzle assembly  284  is mounted on the seed discharge tube holder  286 , which is carried on a discharge actuator  288 . The discharge actuator is mounted on the post  68 . Filters  290  are mounted on the outlets of chip nozzle assembly, to prevent samples being discharged from contaminating the other compartments. 
     In operation, a plurality of seeds is deposited in the hopper  70 . The seed feed mechanism  74  conveys an individual seed to the sampling station  72 . At the sampling station, a sample of material is removed from the seed in a manner that minimizes the impact to the viability of the seed. 
     The sample is removed from the sampling station  72  by the sample conveyor  34 . The venturi device  126  creates an air flow in the tube  120  toward the outlet end  124 . The sample material is drawn into the tube and toward the compartment of the sample tray aligned with outlet end  124  of the tube  120 . The separator  128  separates the sample from the air stream carrying it, and allows the sample to drop into the compartment. In some embodiments, the sample may be distributed to two or more compartments in the sample tray, in which case the two-dimensional translation mechanism  30  is operated to bring one or more additional compartments into alignment with the outlet  124 . It is possible to accurately coordinate the movement of the sample trays with the operation of the sampling station  72  so that samples from different portions of the seed, and in particular different depths of the seed, can be delivered to separate compartments in the sample tray. 
     After the sampling from the seed is completed, the seed conveyor  32  is operated to remove the seed from the sampling station. The first venturi device  106  is operated to create an air flow in the tube and draw a seed from the sampling station  72  into the tube  100 . The second venturi device  108  is then operated to create an air flow in the opposite direction, thereby slowing the seed down to reduce damage to the seed as it exits the outlet end  104  of the tube  100  and is delivered to a compartment in the seed tray  92 . The second venturi  108  stops the movement of the seed, allowing it to drop under gravity to its compartment on a tray  90 . The operation of the first and second venturis  106  and  108  can be timed, or they can be triggered by position sensors monitoring the tube  100 . 
     An embodiment of a high throughput system for determining the fatty acid characteristics of a seed is indicated generally as  500  in  FIGS. 13-26 . As shown in  FIGS. 1 and 2 , the seed sampler system  500  comprises a sampling station  502 , a sample handling station  504 , a seed handling station  506 , and means for analyzing a mixture of fatty acid esters (not shown). It is desirable, but not essential, that the seed sampler system  500  fit on one or more wheeled carts that can pass though conventional doorways, so that the system can be conveniently transported. In this preferred embodiment, the seed sampling station  502  is mounted on a cart  508 , the sample handling station is mounted on a cart  510 , the seed handling station is mounted on a cart  512 , and the means for analysis is mounted on a cart (not shown). 
     The seed sampling station  502  comprises a seed feeder  514  and a seed chipper  516 . A plurality of columns  518  extend vertically upwardly from the surface  520  of the cart  508 . A platform  522  is mounted on top of columns  518  and supports the seed chipper  514 . Two L-brackets  524  extend horizontally from the columns  518 , and support a platform  526 . A stage  528  is mounted on the platform  526  by a plurality of posts  530  and supports the seed feeder  514 . 
     A plurality of pillars  532  extend upwardly from the plate  522 . A plate  534  is mounted on the pillars  532 . A plurality of posts  536  depend from the plate  534 , and support a shelf  538 . 
     As shown in  FIGS. 13 ,  14 ,  15  and  16 , the seed feeder  514  comprises a hopper  550 , with a shaped surface adapted to feed seeds deposited into the hopper toward a separating wheel  552  (see also  FIGS. 23A through 23C ). The separating wheel  552  is mounted for rotation in a vertical plane adjacent the hopper  550 , and as a plurality of spaced recesses  554  each having an opening  556  therein communicating with a vacuum system (not shown). The wheel  552  is advanced with an indexing motor  560 . Individual seeds are picked up by the recesses  554  in the wheel  552  and held in the recesses by suction from the vacuum system via openings  556 . A wiper  562  wipes individual seeds form the recesses  554 , allow them to drop through a guide  564  into an opening in a distributor  566 . 
     As shown in  FIGS. 24-26 , the distributor  566  comprises a shaft  568  having a plurality (six in the preferred embodiment) passages  570  extending transversely therethrough. Sleeves  572  and  574  are slidably mounted over each end of the shaft  568  to translate between first (inboard) and second (outboard) positions. The sleeves  572  and  574  have a plurality of pairs of aligned openings  576  and  578  therein. The openings  576  are elongate, and the openings  576  and  578  are sized and arranged so that when the sleeves  572  and  574  are in their first (inboard) position (on the left side in  FIG. 24 ), a portion of the elongate openings  576  is aligned with a passage  570  in the shaft  568 , and when the sleeves are in their second (outboard) positions a portion of the elongate openings  576  and the second openings  578  are aligned with the passage (on the right side in  FIG. 24 ). An actuator  580  selectively slides the sleeves  572  and  574  between their first and second positions. 
     The distributor  566  is mounted by a bracket  582  on the carriage  584  of a linear actuator  586 , to translate relative to the guide  564 , successively bringing each of the passages  570  in the shaft  568  into alignment with the guide  564  so that a seed can be deposited therein. A seed sensor (not shown) can be mounted adjacent the guide  564  to confirm that a seed is deposited in each passage  570 . A plurality of air nozzles  590  are mounted on the stage  528 , and are aligned with the passages  570  when the distributor  566  is moved to its dispensing position by the actuator  586 . A tube  592  is aligned with each passage  570 , and each tube connects to one of a plurality of seed sampling stations  600  in the seed chipper  516 . The sleeves  572  and  574  are translated allowing the seeds in the passages  570  to drop into tubes  592 . One of the nozzles  590  is aligned with each of the passages  570 , and is actuated to facilitate the movement of the seeds from the passages  570  through the tubes  592  to their respective seed sampling stations  600 . 
     There is preferably a port  596  through the hopper  550  that aligns with the opening  556  in each recess  554  as the wheel  552  turns. The port  596  can be connected to a vacuum to draw any dirt or pieces of seed husks or seed that might clog the openings  556  in the recesses  554 , and impair the ability to of the wheel  552  to select individual seeds from the hopper  550 . 
     The seed chipper  516  comprises at least one, and in this preferred embodiment six, sampling stations  600 . Each seed sampling station  600  removes a sample of material from a seed delivered to it. In this preferred embodiment the sampling stations  600  are arranged or ganged in two groups of three, but the number and arrangement of the sampling stations could vary. The sample handling station  504  receives tissue samples removed from a seed and transported away from each sampling station  600 . Similarly, the seed handling station  506  receives a seed after the sample has been removed from the seed, and the seed is transported from the sampling station  600 . 
     Each seed sampling station  600  has an inlet collar  602  connected to the tube  590 , that opens to a chamber  604 . The bottom surface of the chamber  604  is formed by the end of a rod  606  of actuator  608 . The surface of the bottom is below the inlet collar  602  to ensure that the entire seed drops into the chamber  604  and is not caught in a position only partly in the chamber. A vent  610  may be positioned opposite from the inlet collar  602  to allow air from air nozzles  590  to escape. The vent  610  can be covered with a mesh grille  612  to prevent the seed form escaping the chamber  604  and to cushion the seed as it is delivered into the chamber. 
     This rod  606  lifts a seed out of the chamber  604  and into a seed-receiving recess  614  in the underside of a seed sampling plate  616 . The sampling plate  616  has a sampling opening  618  through which a seed in the seed-receiving recess  614  protrudes. A sampling groove  620  is formed in the top surface of the sampling plate  616  such that a portion of a seed in the recess  614  protrudes into the groove. The sampling plate also has laterally oriented openings  622  and  624  therein aligned with the seed-receiving recess  614 . When the rod  606  lifts a seed delivered to the sampling station  600  into the recess  614  in the plate  616 , fingers  626  and  628  extend transversely through the openings  622  and  624  and are operated by actuator  630  to engage and compress the seed. It has been discovered that compressing at least certain types of seeds during the sampling process can improve viability of the seeds after sampling. For seeds such as soybean seeds, it has been found that a compressive pressure enhances seed viability, and that compressive pressure of between about 2.5 and about 5 pounds is sufficient to enhance viability. 
     A sampling broach  650  having a plurality of cutting edges  652  reciprocates in the groove  620  so that the cutting edges  652  can scrape a sample from a seed being held in the recess  614  by the rod  606  and the fingers  626  and  628 . The cutting edges  652  are preferably parallel, and oriented an oblique angle less than 90° relative the direction of travel of the broach. It is desirable, but not essential, that the cutting edges  652  be angled sufficiently that one edge remains in contact with the seed at all time. Angling the cutting edges allows the next blade to establish contact with the seed before the current blade loses contact with the seed. In the preferred embodiment the cutting edges are oriented at an angle of about 60°, although this angle will depend somewhat upon the width of the broach. The width of the broach can also be an important to preserving seed viability after sampling, and will vary depending upon the type of seed and its moisture content. 
     The cutting edges  652  are staggered, each cutting progressively deeper than the previous. The amount of sample material and the depth of the cut can be controlled by controlling the advancement of the broach  650 . For smaller samples and shallower depths of cut, the stroke of the broach  650  is shorter, and for larger samples or deeper depths of cut, the stroke of the broach is longer. For partial stokes, tissue from the seed may be trapped between edges  652 . The broach  650  can be advanced and refracted to help release all of the sample. For example, after the seed is released, the broach may be advanced and retracted to help remove seed tissue trapped between the cutting edges. The full range of travel of the broach  650  is shown in  FIGS. 19A and 19B . 
     The sampling broach  650  is preferably driven by a linear actuator  654 . In the preferred embodiment, three broaches  650  are driven by a single actuator  654 . Using a single actuator to operate multiple broaches saves space and is more economical. 
     A sample transport system  656  comprising a conduit  658  having an inlet  660  communicating a passage  662  that opens to the sampling opening  618  and the groove  620  in the sampling plate  616  removes tissue samples made by the action of the cutting edges  652  of the sampling broach  650 . The conduit  658  transports the sample to outlet  664  where it is deposited in a unique sample holder in the sample handling station  504 . This sample holder may be, for example, a well  666  in a tray  668  mounted on a x-y indexing table  670  on cart  510 , so that the relationship between samples and their respective seeds can be determined. The sample transport system  656  includes an air jet  672  which induces air flow through the conduit  658  to move the sample through the conduit. 
     A second sampling mechanism in mounted on the linear actuator  654  and moves with the broach  650 . The second sampling mechanism comprises a coring device  674  having a coring tool  676  for taking a plug sample of the seed from the kerf made by the broach  650 . This tissue in this sample is from a deeper location than the tissue scraped by the broach  650 , and provides different information. In some embodiments the material removed by the broach  650  might simply be discarded, and only the sample taken with the coring device  674  retained. In some embodiments both samples may be retained and separately stored for separate testing. In still other embodiments the only sample is the sample removed by the broach  650 . In embodiments without the second sampling mechanism, the coring device  674  and coring tool  676  can be replaced with an actuator with a simple push rod that extends through the sampling opening  618  to help push a seed in the recess  614 . 
     A seed transport system  680  having an inlet  682  adjacent recess  614  for drawing in seeds after they are released by the fingers  626  and  628  and the rod  606  lowers the seed after the sampling operation. The seed transport system  680  transports the seeds to a unique seed holder in the seed handling station  506  on the cart  512 . This seed holder may be, for example, a well  684  in a tray  686  mounted on an x-y indexing table  688  on cart  612 , so that the relationship between samples and their respective seeds can be determined. The seed transport mechanism  680  includes an air jet  690  which induces air flow through the conduit  680  to move the sample through the conduit. 
     In operation, a plurality of seeds, oil seeds such as soybeans, corn, maize, canola, rapeseed, sunflower, peanut, safflower, palm, cotton, etc., are dumped into the hopper  550  of the sampling system  500 . These seeds flow under gravity toward the disk  552 , suction through the ports  556  hold one seed in each cavity  554 . As the disk  552  is rotated by the indexing motor  560 , individual seeds are wiped from the disk by the wiper  562 , and fall under gravity through the guide  564  to the outlet. The linear actuator  586  moves the distributor  566  so that each passage  570  of the distributor aligns with the guide  564  to load one seed through the opening  576  and into passage  570 . When all of the passages  570  in the distributor  566  are full, the linear actuator  586  moves the distributor into position to load its seeds into sampling stations  600  in the seed chipper  516 . The sleeves  572  and  574  are moved by actuator  580 , which aligns the openings  578  with the passages  570 , allowing the seeds in the passages  570  to fall into the tubes  592  that lead to the sampling units  600 . The nozzles  590  provide a blast of air that helps urge the seeds from the passages  570  through the tubes  592  to the chambers  604  in the sampling units  600 . 
     Preferably all of the passage  570  are loaded in series and discharge their seeds simultaneously to the sampling units  600 , but the distributor could be programmed to operate in some other manner. Once the seeds arrive in the sampling stations  600 , the rod  606  lifts the seed into the recess  614  in the underside of the plate  616 . The recess  614  may be sized and shaped to help optimally orient the seed. In the recess  614 , a portion of the seed protrudes through the sampling hole  618  and into the groove  620 . The broach  650  is translated in the groove  620 , allowing its cutting edges  652  to remove material from the portion of the seed protruding into the groove  620 , and forming a small kerf in the seed. As the broach  650  removes material, the sample transport system  656  draws the sample material through passage  662  and into the inlet  660 . The sample travels in conduit  658  away from the sampling station  600  to a sample storage location, such as a well  666  in a sample tray  668 . A second sample can be taken by the coring tool  676  of sampling device  674  through the opening  618  in the sampling plate  616 . After the sampling is completed, the rod  606  retracts, and as the seed drops the sampled-seed transport system  680  transports the sampled seed to a seed storage location, such as a well  684  in a seed tray  686 . 
     The indexing tables  670  and  688  move to align different wells with the outlets of the sample transport system  656  and the seed transport system  680 , and the sample process is repeated. When all of the wells  666  in a sample tray  668 , the samples in the sample tray can be tested, and the seeds in the corresponding seed tray  686  can be selected based upon the results of the testing of samples. The sampling preferably does not substantially adversely affect the viability of the seeds. 
     EXAMPLES 
     The following examples are merely illustrative, and not limiting to this disclosure in any way. 
     Example 1 
     This example demonstrates the use of the screening methods of the present invention in a program for selection and bulking of Low Linolenic Acid soybeans. 
     Soybean is the most valuable legume crop, with many nutritional and industrial uses due to its unique chemical composition. Soybean seeds are an important source of vegetable oil, which is used in food products throughout the world. The relatively high level (usually about 8%) of linolenic acid (18:3) in soybean oil reduces its stability and flavor. Hydrogenation of soybean oil is used to lower the level of linolenic acid (18:3) and improve both stability and flavor of soybean oils. However, hydrogenation results in the production of trans fatty acids, which increases the risk for coronary heart disease when consumed. The development of low linolenic acid soybeans has been complicated by the quantitative nature of the trait. The low linolenic acid soybean varieties that have been developed have been found to yield poorly, limiting their usefulness in most commercial settings. Developing a product with commercially significant seed yield is a high priority in most soybean cultivar development programs. 
     Seed tissue samples (about 5 mg each) were collected from both regular soybean varieties and low linolenic acid soybean varieties and transferred to the individual wells of a 96-well microtiter plate. The samples were then wetted with toluene to extract and transmethylate oil in the samples to produce a mixture of fatty acid methyl esters. The mixture of fatty acid methyl esters were then removed from the wells of the microtiter plate and analyzed on a gas chromatograph. 
     The chromatograph (Supelco Omegawax 320 capillary column using flame ionization detection) was programmed to run in “fast” mode wherein a fast temperature ramp produces a chromatogram in 3.6 minutes. An example of a chromatogram of fatty acid methyl esters for a normal soybean analyzed in the experiment is shown in  FIG. 29 . An example of a chromatogram of fatty acid methyl esters obtained from a low linolenic acid soybean in accordance with this experiment is shown in  FIG. 30 . 
     The average fatty acid characteristics for regular soybeans analyzed in this experiment are shown in Table 1. 
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Normal Soybeans 
               
             
          
           
               
                   
                 Fatty Acid (% relative) 
                 Average 
               
               
                   
                   
               
               
                   
                 C 16  Palmitic acid 
                 12.8 ± 0.3 
               
               
                   
                 C 18  Steric acid 
                  4.2 ± 0.1 
               
               
                   
                 C 18: 1n9  Oleic acid 
                 16.1 ± 1.6 
               
               
                   
                 C 18: 2n6  Linolenic acid 
                 53.5 ± 0.9 
               
               
                   
                 C 18: 3  Linolenic acid 
                  8.8 ± 0.8 
               
               
                   
                   
               
             
          
         
       
     
     The average fatty acid characteristics for a low linolenic acid soybeans analyzed in this experiment are shown in Table 2. 
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Low Linolenic Soybeans 
               
             
          
           
               
                   
                 Fatty Acid (% relative) 
                 Average 
               
               
                   
                   
               
               
                   
                 C 16  Palmitic acid 
                 10.4 ± 0.3 
               
               
                   
                 C 18  Steric acid 
                  4.6 ± 0.4 
               
               
                   
                 C 18: 1n9  Oleic acid 
                 19.3 ± 0.9 
               
               
                   
                 C 18: 2n6  Linolenic acid 
                 59.1 ± 1.0 
               
               
                   
                 C 18: 3  Linolenic acid 
                  3.0 ± 0.3 
               
               
                   
                   
               
             
          
         
       
     
     The selected seed having the desired fatty acid characteristics may be bulked or kept separate depending on the breeding objectives. These seeds could be planted in the field with appropriate field identification. Several methods of preserving single seed identity can be used while transferring seed from the lab to the field. Methods include transferring selected individuals to horticultural seed tape that could also include radio frequency identification to aid in the identification of the individual genotyped seed. Other methods would be to use an indexing tray, plant seeds in peat pots and then transplant them, or hand plant from individual seed packets. 
     Example 2 
     This example demonstrates the use of the screening methods of the present invention in a program for selecting and bulking of Stearidonic Acid (SDA) soybeans. 
     Tissue samples were collected from soybean varieties identified as 0% SDA, 15% SDA, 20% SDA, and 30% SDA. The tissue samples were contacted with solvent to produce a mixture of fatty acid esters and the fatty acid esters were then separated and analyzed using fast gas chromatography as described in Example 1. The fatty acid profiles of the SDA soybeans are shown in Table 3. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Fast GC Method and SDA Soybeans 
               
             
          
           
               
                   
                 0% 
                 15% 
                 20% 
                 30% 
               
               
                 Fatty acid (% relative) 
                 SDA 
                 SDA 
                 SDA 
                 SDA 
               
               
                   
               
             
          
           
               
                 C 14  Myristic acid 
                 0 
                 0.3 
                 0.3 
                 0.3 
               
               
                 C 16  Palmitic acid 
                 11.9 
                 12.5 
                 12.7 
                 13.1 
               
               
                 C 18  Steric acid 
                 3.8 
                 3.7 
                 3.7 
                 3.7 
               
               
                 C 18: 1 n 9  Oleic acid 
                 20.3 
                 15 
                 17.1 
                 15.3 
               
               
                 C 18: 2 n 6  Linoleic acid 
                 50.8 
                 32 
                 28.2 
                 17 
               
               
                 C 18: 3 n 6  gamma Linolenic 
                 — 
                 3.8 
                 4.8 
                 4.6 
               
               
                 C 18: 3  Linolenic acid 
                 7.7 
                 11.1 
                 10.5 
                 12.2 
               
               
                 C 18: 4 n 3  Octadecatetraenoic 
                 — 
                 13 
                 16 
                 26.8 
               
               
                 C 20  Arachidonic acid 
                 0.6 
                 0.8 
                 0.6 
                 0.7 
               
               
                 C 20: 1 n 9  Eicosenoic acid 
                 0.2 
                 0.4 
                 0.3 
                 0.4 
               
               
                 C 22  Behenic acid 
                 0.3 
                 0.3 
                 0.3 
                 0.4 
               
               
                 C 24  Lignoceric acid 
                 0 
                 0.1 
                 0.1 
                 0.1 
               
               
                   
               
             
          
         
       
     
     Example 3 
     This example demonstrates the use of the screening methods of the present invention in a program for selection and bulking of High Stearic Acid soybeans. 
     Tissue samples were collected from soybean varieties identified as high stearic acid soybeans. The tissue samples were contacted with solvent to produce a mixture of fatty acid esters and the fatty acid esters were then separated and analyzed using fast gas chromatography as described in Example 1. The fatty acid profiles of the high stearic acid soybeans are shown in Table 4. 
     
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 High Stearic Acid Soybeans 
               
             
          
           
               
                   
                 Fatty acid (% relative) 
                 Fast GC method 
               
               
                   
                   
               
             
          
           
               
                   
                 C 14  Myristic acid 
                 0 
               
               
                   
                 C 16  Palmitic acid 
                 8.9 
               
               
                   
                 C 18  Steric acid 
                 20.3 
               
               
                   
                 C 18: 1 n 9  Oleic acid 
                 21.4 
               
               
                   
                 C 18: 2 n 6  Linoleic acid 
                 37.8 
               
               
                   
                 C 18: 3  Linolenic acid 
                 3.1 
               
               
                   
                 C 20  Arachidonic acid 
                 1.8 
               
               
                   
                 C 20: 1 n 9  Eicosenoic acid 
                 0.1 
               
               
                   
                 C 22  Behenic acid 
                 1.0 
               
               
                   
                 C 24  Lignoceric acid 
                 0.2 
               
               
                   
                   
               
             
          
         
       
     
     Example 4 
     This example demonstrates the use of the screening methods of the present invention in a program for screening rapeseed. 
     Tissue samples collected from rapeseed were contacted with toluene to produce a mixture of fatty acid esters. The fatty acid esters were then separated and analyzed using fast gas chromatography as described in Example 1. The samples were screened and identified as follows: (1) conventional rapeseed (i.e., having an erucic acid content less than about 2%); (2) having an erucic acid content greater than about 2%; (3) having an erucic acid content of greater than about 45%; (4) having an erucic acid content of greater than 45% and a linolenic acid content of less than about 3.5%; (5) having a linolenic acid content of less than about 3.5%; (6) having an oleic acid content of greater than about 70%; (7) having less than about 7% saturated fat; (8) having less than about 6% saturated fat; (9) having less than about 5% saturated fat; (10) having an oleic acid content of greater than about 70% and a linolenic acid content of less than about 3.5%; and (11) having an oleic acid content of greater than about 70%, a linolenic acid content of less than about 3.5%, and less than about 7% saturated fat. 
     Example 5 
     This example demonstrates the use of the screening methods of the present invention in a program for screening sunflower. 
     Tissue samples collected from sunflower seeds were contacted with toluene to produce a mixture of fatty acid esters. The fatty acid esters were then separated and analyzed using fast gas chromatography as described in Example 1. The samples were screened and identified as follows: (1) an oleic acid content of from about 40% to about 70%, (2) an oleic acid content of greater than about 70%, (3) a stearic acid content of greater than about 6%, (4) a saturated fat content of less than about 8%, (5) an oleic acid content of greater than about 70% and a saturated fat content of less than about 8%, and (6) an oleic acid content of greater than about 70%, a stearic acid content of greater than about 6%, and a saturated fat content of less than about 8%.

Technology Classification (CPC): 6