U.S. patent application number 11/510771 was filed with the patent office on 2007-03-01 for high throughput screening of fatty acid composition.
Invention is credited to Pradip K. Das, Kevin L. Deppermann, Luis Jurado, Dutt V. Vinjamoori.
Application Number | 20070048872 11/510771 |
Document ID | / |
Family ID | 37613895 |
Filed Date | 2007-03-01 |
United States Patent
Application |
20070048872 |
Kind Code |
A1 |
Deppermann; Kevin L. ; et
al. |
March 1, 2007 |
High throughput screening of fatty acid composition
Abstract
A method for the high throughput screening of fatty acid
characteristics in seeds is provided. The method comprises feeding
seeds individually to a sampling station; removing a sample from
the seed in the sampling station; conveying the sample to a
compartment in a sample tray; converting extracted oil from the
sample in the sample tray to form a mixture of fatty acid methyl
esters; and analyzing the mixture of fatty acid methyl esters from
the sample to determine the fatty acid profile of the corresponding
seed.
Inventors: |
Deppermann; Kevin L.; (St.
Charles, MO) ; Jurado; Luis; (St. Louis, MO) ;
Vinjamoori; Dutt V.; (Chesterfield, MO) ; Das; Pradip
K.; (Olivette, MO) |
Correspondence
Address: |
HARNESS, DICKEY, & PIERCE, P.L.C
7700 BONHOMME, STE 400
ST. LOUIS
MO
63105
US
|
Family ID: |
37613895 |
Appl. No.: |
11/510771 |
Filed: |
August 25, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60711775 |
Aug 26, 2005 |
|
|
|
Current U.S.
Class: |
436/71 |
Current CPC
Class: |
B07C 2501/0081 20130101;
C11B 1/10 20130101; G01N 33/03 20130101; G01N 33/92 20130101; G01N
33/5097 20130101 |
Class at
Publication: |
436/071 |
International
Class: |
G01N 33/92 20060101
G01N033/92 |
Claims
1. An automated method for determining the fatty acid composition
of a plurality of seeds, the method comprising: 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; converting 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.
2. The method of claim 1 wherein the steps of extracting oil and
converting extracted oil comprise contacting the sample in the
sample tray with a solvent to form the mixture of fatty acid
esters.
3. The method of claim 2, wherein the solvent is selected from the
group consisting of hexane, benzene, tetrahydrofuran, dimethyl
sulfoxide, trimethylsulfonium hydroxide, petroleum ether, methylene
chloride, and toluene.
4. The method of claim 2, wherein the solvent is toluene.
5. The method of claim 1, wherein the step of analyzing comprises
separating and detecting the mixture of fatty acid esters using gas
chromatography.
6. The method of claim 1, wherein the sample is scraped from the
seed without affecting the germination viability of the seed.
7. The method of claim 1, wherein the sample tray comprises a
96-well microtiter plate.
8. The method of claim 1, wherein the sample tray comprises a
384-well microtiter plate.
9. A method for high throughput screening of oil seeds, the method
comprising: 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 seed; and selecting seed based on
the presence or absence of a desired fatty acid characteristic.
10. The method of claim 9, wherein the step of analyzing comprises
separating and detecting the fatty acid methyl esters using gas
chromatography.
11. The method of claim 9, wherein 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 toluene.
12. The method of claim 9, wherein the method further comprises
collecting the tissue samples without affecting the germination
viability of the seed.
13. The method of claim 9, wherein the sample tray comprises at
least 96 individual compartments.
14. The method of claim 9, wherein the oil seeds are selected from
the group consisting of soybean, corn, canola, rapeseed, sunflower,
peanut, safflower, palm and cotton.
15. The method of claim 14, wherein the oil seed is rapeseed and
the desired fatty acid characteristic is an erucic acid content of
greater than about 2%.
16. The method of claim 14, wherein the seed is rapeseed and the
desired fatty acid characteristic is an erucic acid content of
greater than about 45%.
17. The method of claim 14, wherein the seed is rapeseed and the
desired fatty acid characteristic is a linolenic acid content of
less than about 3.5%.
18. The method of claim 14, wherein the seed is rapeseed and the
desired fatty acid characteristic is an oleic acid content of
greater than about 70%.
19. The method of claim 14, wherein the seed is rapeseed and the
desired fatty acid characteristic is a saturated fat content of
less than about 7%.
20. The method of claim 14, wherein the seed is rapeseed and the
desired fatty acid characteristic is a saturated fat content of
less than about 6%.
21. The method of claim 14, wherein the seed is rapeseed and the
desired fatty acid characteristic is a saturated fat content of
less than about 5%.
22. The method of claim 14, wherein the seed is rapeseed and the
desired fatty acid characteristic is an oleic acid content of
greater than about 70% and a linolenic acid content of less than
about 3.5%.
23. The method of claim 14, wherein the seed is rapeseed and the
desired fatty acid characteristic is an oleic acid content of
greater than about 70%, a linolenic acid content of less than about
3.5%, and a saturated fat content of less than about 7%.
24. The method of claim 14, wherein the seed is rapeseed and the
desired fatty acid characteristic is an erucic acid content of
greater than about 45% and a linolenic acid content of less than
about 3.5%.
25. The method of claim 14, wherein the seed is sunflower and the
desired fatty acid characteristic is an oleic acid content of from
about 40% to about 70%.
26. The method of claim 14, wherein the seed is sunflower and the
desired fatty acid characteristic is an oleic acid content of
greater than about 70%.
27. The method of claim 14, wherein the seed is sunflower and the
desired fatty acid characteristic is a stearic acid content of
greater than about 6%.
28. The method of claim 14, wherein the seed is sunflower and the
desired fatty acid characteristic is a saturated fat content of
less than about 8%.
29. The method of claim 14, wherein the seed is sunflower and the
desired fatty acid characteristic is an oleic acid content of
greater than about 70% and a saturated fat content of less than
about 8%.
30. The method of claim 14, wherein the seed is sunflower and the
desired fatty acid characteristic is 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%.
31. The method of claim 14, wherein the seed is soybean and the
desired fatty acid characteristic is a linolenic acid content of
less than about 8%.
32. The method of claim 9, wherein the method further comprises
cultivating plants from the selected seed; and harvesting seeds
from the cultivated plants.
33. A method of bulking up a quantity of seed having a desired
fatty acid characteristic, the method comprising: (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.
34. The method of claim 33, wherein the solvent is selected from
the group consisting of hexane, benzene, tetrahydrofuran, dimethyl
sulfoxide, trimethylsulfonium hydroxide, petroleum ether, methylene
chloride, and toluene.
35. The method of claim 34, wherein the solvent is toluene.
36. The method of claim 33, wherein the step of analyzing comprises
separating and detecting the mixture of fatty acid esters using gas
chromatography.
37. The method of claim 33, wherein 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.
38. The method of claim 33, wherein the seeds are oil seeds
selected from the group consisting of soybean, corn, canola,
rapeseed, sunflower, peanut, safflower, palm and cotton.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Application Ser. No. 60/711,755, filed Aug. 26, 2005, the entire
disclosure of which is hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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.
[0004] 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
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] These and other features and advantages will be in part
apparent, and in part pointed out hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a perspective view of one embodiment of an
automated seed sampler system for use according to the principles
of this invention;
[0012] FIG. 2 is an enlarged perspective view of the seed sampler
assembly of the seed sampler system;
[0013] FIG. 3 is an enlarged perspective view of the hopper and
seed feeding mechanism of the seed sampler assembly;
[0014] FIG. 4 is a perspective view of the broach for scraping
samples from the seeds;
[0015] FIG. 5 is a perspective view of the slide for driving the
broach of the piston actuator from the seed feeding mechanism;
[0016] FIG. 6 is a perspective view of the piston in the feed
mechanism of the hopper;
[0017] FIG. 7 is a perspective view of the stage with a plurality
of seed trays and sample trays mounted thereon;
[0018] FIG. 8 is a perspective view of the two-dimensional
translation mechanism;
[0019] FIG. 9 is a perspective view of the inlet of the seed
conveyor;
[0020] FIG. 10 is a perspective view of the outlet of the seed
conveyor;
[0021] FIG. 11 is a perspective view of the outlet of the sample
conveyor;
[0022] FIG. 12 is a perspective view of the air multiplier used in
the seed and sample conveyors;
[0023] FIG. 13 is a top plan view of a high throughput seed sampler
system for use in accordance with the principles of this
invention;
[0024] FIG. 14 is a side elevation view of the high throughput seed
sampler device;
[0025] FIG. 15 is a front perspective view of the seed sampler
system;
[0026] FIG. 16 is a rear perspective view of the seed sampler
system;
[0027] FIG. 17 is a perspective view of the sampling station of the
high throughput seed sampler device;
[0028] 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;
[0029] 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;
[0030] FIG. 19A is a side elevation view of the seed sampling
station, with the broach in its retracted position;
[0031] FIG. 19B is a side elevation view of the seed sampling
station, with the broach in its extended position;
[0032] FIG. 20 is a longitudinal cross-sectional view of the seed
sampling station;
[0033] FIG. 21 is a front end elevation view of the seed sampling
station;
[0034] FIG. 22 is a transverse cross-sectional view of the seed
sampling station;
[0035] FIG. 23A is a side elevation view of the seed selecting
wheel;
[0036] FIG. 23B is an exploded view of the seed selecting
wheel;
[0037] FIG. 23C is a vertical cross sectional view of the seed
selecting wheel;
[0038] FIG. 24 is a front elevation view of the feeding
mechanism;
[0039] FIG. 25 is a side elevation view of the feeding
mechanism;
[0040] FIG. 26A is a perspective view of the feeding mechanism;
[0041] FIG. 26B is a side elevation view of the feeding
mechanism;
[0042] FIG. 26C is a longitudinal cross-sectional view of the
feeding mechanism, taken along the plane of line 26C-26C;
[0043] FIG. 26D is a bottom plan view of the feeding mechanism;
[0044] FIG. 27A is a vertical longitudinal cross-sectional view of
the sampling mechanism;
[0045] FIG. 27B is an enlarged partial vertical cross sectional
view of the sampling mechanism as shown in FIG. 27A;
[0046] FIG. 28A is a vertical transverse cross-sectional view of
the sampling mechanism;
[0047] FIG. 28B is an enlarged partial cross-sectional view of the
sampling mechanism as shown in FIG. 28A;
[0048] FIG. 29 is a chromatogram of fatty acid esters obtained from
a normal soybean in accordance with the method described in Example
1; and
[0049] 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.
[0050] Corresponding reference numerals indicate corresponding
parts throughout the several views of the drawings.
DETAILED DESCRIPTION
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.sub.2 population for fatty acid characteristics,
all individuals with the desired fatty acid profile may be bulked
and planted in the breeding nursery.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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
[0069] 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.
[0070] 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.
[0071] 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).
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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).
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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.
[0099] 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.degree. 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.degree., 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.
[0100] 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
retracted 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.
[0101] 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] 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
[0108] The following examples are merely illustrative, and not
limiting to this disclosure in any way.
Example 1
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] The average fatty acid characteristics for regular soybeans
analyzed in this experiment are shown in Table 1. TABLE-US-00001
TABLE 1 Normal Soybeans Fatty Acid (% relative) Average C.sub.16
Palmitic acid 12.8 .+-. 0.3 C.sub.18 Steric acid 4.2 .+-. 0.1
C.sub.18:1n9 Oleic acid 16.1 .+-. 1.6 C.sub.18:2n6 Linolenic acid
53.5 .+-. 0.9 C.sub.18:3 Linolenic acid 8.8 .+-. 0.8
[0114] The average fatty acid characteristics for a low linolenic
acid soybeans analyzed in this experiment are shown in Table 2.
TABLE-US-00002 TABLE 2 Low Linolenic Soybeans Fatty Acid (%
relative) Average C.sub.16 Palmitic acid 10.4 .+-. 0.3 C.sub.18
Steric acid 4.6 .+-. 0.4 C.sub.18:1n9 Oleic acid 19.3 .+-. 0.9
C.sub.18:2n6 Linolenic acid 59.1 .+-. 1.0 C.sub.18:3 Linolenic acid
3.0 .+-. 0.3
[0115] 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
[0116] 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.
[0117] 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-US-00003 TABLE 3 Fast GC Method and SDA Soybeans Fatty acid
(% relative) 0% SDA 15% SDA 20% SDA 30% SDA C.sub.14 Myristic acid
0 0.3 0.3 0.3 C.sub.16 Palmitic acid 11.9 12.5 12.7 13.1 C.sub.18
Steric acid 3.8 3.7 3.7 3.7 C.sub.18:1n9 Oleic acid 20.3 15 17.1
15.3 C.sub.18:2n6 Linoleic acid 50.8 32 28.2 17 C.sub.18:3n6 gamma
Linolenic -- 3.8 4.8 4.6 C.sub.18:3 Linolenic acid 7.7 11.1 10.5
12.2 C.sub.18:4n3 -- 13 16 26.8 Octadecatetraenoic C.sub.20
Arachidonic acid 0.6 0.8 0.6 0.7 C.sub.20:1n9 Eicosenoic acid 0.2
0.4 0.3 0.4 C.sub.22 Behenic acid 0.3 0.3 0.3 0.4 C.sub.24
Lignoceric acid 0 0.1 0.1 0.1
Example 3
[0118] 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.
[0119] 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-US-00004 TABLE 4 High Stearic Acid Soybeans Fatty acid (%
relative) Fast GC method C.sub.14 Myristic acid 0 C.sub.16 Palmitic
acid 8.9 C.sub.18 Steric acid 20.3 C.sub.18:1n9 Oleic acid 21.4
C.sub.18:2n6 Linoleic acid 37.8 C.sub.18:3 Linolenic acid 3.1
C.sub.20 Arachidonic acid 1.8 C.sub.20:1n9 Eicosenoic acid 0.1
C.sub.22 Behenic acid 1.0 C.sub.24 Lignoceric acid 0.2
Example 4
[0120] This example demonstrates the use of the screening methods
of the present invention in a program for screening rapeseed.
[0121] 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
[0122] This example demonstrates the use of the screening methods
of the present invention in a program for screening sunflower.
[0123] 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%.
* * * * *