Note: Descriptions are shown in the official language in which they were submitted.
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Methods for Determining Oil in Seeds
Cross-reference to Related Application
This application claims priority to Provisional Patent Application U.S. Serial
No.
60/156,287 entitled "Method for Determining Total Oil in Seeds," which was
filed September
27, 1999.
Field of the Invention
The present invention relates to methods for analyzing agricultural products.
More
particularly, the present invention relates to methods for analysis of the oil
content of one or
l0 more seeds.
Background of the Invention
The improvement of techniques used for analysis of agricultural products for
desired
traits has long been a goal. Several methods have conventionally been used to
analyze a
15 sample for the presence of a specific trait. Quantitation of oil content of
seeds is often
performed with conventional methods, such as near infrared analysis (I~IIR),
nuclear magnetic
resonance imaging (NMR), soxhlet extraction, accelerated solvent extraction
(ASE),
microwave extraction, and super critical fluid extraction. These conventional
methods,
however, are often not able to accurately discern the relative or absolute
levels of oil in very
2o small seed samples.
During the past decade, near infrared (NIR) spectroscopy has become a standard
method for screening seed samples whenever the sample of interest has been
amenable to this
technique. Samples studied include wheat, maize, soybean, canola, rice,
alfalfa, oat, and
others (see, for example, Massie and Norris, "Spectral Reflectance and
Transmittance
25 Properties of Grain in the Visible and Near Infrared", Transactions of the
ASAE, Winter
Meeting of the American society of Agricultural Engineers, 1965, pp. 598-600,
Archibald et
al. "Development of Short-Wavelength Near-Infrared spectral Imaging for Grain
Color
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Classification," SPIE Vol. 3543, 1998, pp. 189-198, Delwiche, "Single Wheat
Kernel
Analysis by Near-Infrared Transmittance: Protein Content," Analytical
Techniques and
Instrumentation, Vol. 72, 1995, pp. 11-16, Dowell, "Automated Color
Classification of Single
Wheat Kernels Using Visible and Near-Infrared Reflectance," Vol. 75(1), 1998,
pp.142-144,
Orman and Schumann, "Comparison of Near-Infrared Spectroscopy Calibration
Methods for
the Prediction of Protein, Oil, and Starch in Maize Grain," Vol. 39, 1991,
pp.883-886,
Robutti, "Maize Kernel Hardness Estimation in Breeding by Near-Infrared
Transmission
Analysis," Vol. 72(6), 1995, pp.632-636, U.S. Patent No. 5,991,025 to Wright
et al., U.S.
Patent No. 5.751,421 to Wright et al., Daun et al., "Comparison of Three whole
Seed Near-
1o Infrared Analyzers for Measuring Quality Components of Canola Seed", Vol.
71, no. 10,
1994, pp.1063-1068, all of which are herein incorporated by reference in their
entirety).
Nit analysis of single seeds has been reported (see Velasco, et al.,
"Estimation of
Seed Weight, Oil Content and Fatty Acid Composition in Intact Single Seeds of
Rapeseed
(Brassica napus L.) by Rear-Infrared Reflectance Spectroscopy," Euphytica,
Vol. 106, 1999,
pp.79-85, Delwiche, "Single Wheat Kernel Analysis by Near-Infrared
Transmittance: Protein
Content," Analytical Techniques and Instrumentation, Vol. 72, 1995, pp. 11-16,
Dowell,
"Automated Color Classification of Single Wheat Kernels Using Visible and Near-
Infrared
Reflectance," Vol. 75(1), 1998, pp.142-144, Dowell et al., "Automated Single
Wheat Kernel
Quality Measurement Using Near-Infrared Reflectance," ASAE Annual
International
2o Meeting, 1997, paper number 973022, all of which are herein incorporated by
reference in
their entirety). These methods, however, are not sensitive enough to
accurately determine the
oil content of very small seeds, which limits their use. NMR has also been
used to analyze
oil content in seeds (see, for example, Robertson and Morrison, "Analysis of
Oil Content of
Sunflower Seed by Wide-Line NMR," Journal of the American Oil Chemists
Society, 1979,
Vol. 56, 1979, pp. 961-964, which is herein incorporated by reference in its
entirety).
However. this non-destructive technique is also often not suitable for the
analysis of seed oil
when the seed of interest is small.
2
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Other techniques, including soxhlet extraction, accelerated solvent extraction
(ASE),
microwave extraction, and super critical fluid extraction, that are
conventionally used to
determine oil content use gravimetry as the final measurement step (see, for
example, Taylor
et al., "Determination of Oil Content in Oilseeds by Analytical Supercritical
Fluid
Extraction," Vol. 70 (no. 4), 1993, pp. 437-439, which is herein incorporated
by reference in
its entirety). Gravimetry, however. is not suitable for use with small
samples. including small
seeds and seed with little oil content. because oil levels in these samples
can be below the
level of minimum sensitivity for the technique. Further, the use of gravimetry
is time
consuming and is not amenable to high-throughput automation.
to Needed in the art are methods for rapid and accurate analysis of seed
samples, and
particularly small seed samples, that can be used to efficiently analyze the
oil content of
individual seeds and that are amenable to automation. The present invention
provides such
methods.
Summary Of The Invention
The present invention includes and provides a method for determining oil
content of a
seed comprising: extracting oil from a seed using a solvent; evaporating the
solvent in a
stream of gas to form oil particles: directing light into the stream of gas
and the oil particles,
thereby forming reflected light; detecting the reflected light; and,
determining the oil content
based on the reflected light.
2o The present invention includes and provides a method for determining oil
content of a
seed comprising extracting oil from a seed using a solvent; separating the
solvent from the
seed; evaporating the solvent in a stream of gas to form oil particles;
directing light into the
stream of gas and the oil particles. thereby forming reflected light;
detecting the reflected
light; and, determining the oil content based on the reflected light.
The present invention includes and provides a method for determining oil
content of a
seed comprising: disrupting the seed to produce ground seed; extracting oil
from the ground
seed using a solvent; evaporating the solvent in a stream of gas to form oil
particles: directing
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light into the stream of gas and the oil particles, thereby forming reflected
light; detecting
the reflected light; determining the oil content based on the reflected light.
The present invention includes and provides a method for determining oil
content of
an agricultural material, comprising: extracting oil from the material using a
solvent;
evaporating the solvent in a stream of gas to form oil particles; directing
light into the stream
of gas and the oil particles, thereby forming reflected light; detecting the
reflected light; and,
determining the oil content based on the reflected light.
The present invention includes and provides a method for determining oil
content of a
batch seed sample, comprising: extracting oil from the batch seed sample using
a solvent;
1o evaporating the solvent in a stream of gas to form oil particles; directing
light into the stream
of gas and the oil particles, thereby forming reflected light; detecting the
reflected light; and,
determining the oil content based on the reflected light.
The present invention includes and provides a method for selecting a seed
having an
enhanced oil content, comprising: extracting oil from a seed using a solvent;
evaporating
the solvent in a stream of gas to form oil particles; directing light into the
stream of gas and
the oil particles, thereby forming reflected light; detecting the reflected
light;
determining the oil content based on the reflected light; and, selecting a
seed with a
similar genetic background to the seed based on the oil content.
The present invention includes and provides a method of introgressing a trait
into a
plant comprising: extracting oil from a seed using a solvent; evaporating the
solvent in a
stream of gas to form oil particles; directing light into the stream of gas
and the oil particles,
thereby forming reflected light; detecting the reflected light; determining
the oil content based
on the reflected light; selecting a seed with a similar genetic background to
the seed based on
the oil content; growing a fertile plant from the related seed; and, utilizing
the fertile plant as
either a female parent or a male parent in a cross with a second plant.
The present invention includes and provides a method for determining oil
content of a
seed comprising: extracting oil from a seed using a solvent; nebulizing the
solvent and the oil
4
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under high pressure into a device capable of evaporating the solvent;
evaporating the solvent
in a stream of gas in the device to form oil particles; directing light into
the stream of gas and
the oil particles, thereby forming reflected light; detecting the reflected
light; determining the
oil content based on the reflected light.
The present invention includes and provides a method for selecting a seed
having an
enhanced oil content, comprising: a) extracting oil from a seed using a
solvent; b)
evaporating the solvent in a stream of gas to form oil particles; c) directing
light into the
stream of gas and the oil particles, thereby forming reflected light; d)
detecting the reflected
light; e) determining the oil content based on the reflected light; f)
repeating steps a)
1o through e) one or more times, and, g) selecting one or more seeds based on
the oil content.
Description Of The Figures
Figure 1 is a schematic diagram of a cross section of one embodiment of a
system that
is capable of carrying out the methods of the present invention.
15 Figure 2 is plot showing a calibration curve for oil content.
Figure 3 is a chromatograph of extracted soybean oil content.
Figure 4 is a table that compares the results of the present invention with
the
conventional technique of accelerated solvent extraction for soybean.
Figure 5 is a graph that compares the results of the present invention with
the
2o conventional technique of accelerated solvent extraction for sorghum.
Figure 6 is graph that compares the results of the present invention with the
conventional technique of accelerated solvent extraction for maize.
Figure 7 is a graph showing the reproducibility of one embodiment of the
present
invention for maize.
25 Figure 8 is a overlayed chromatograph showing oil content determination for
one,
two, and three Arabidopsis thaliana seeds.
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Detailed Description Of The Invention
Analytical Methods
The present invention provides analytical methods for selecting seeds having a
desired
oil content. In one embodiment, the present invention is a method for
determining oil content
of a single seed comprising: extracting oil from a single seed using a
solvent; evaporating the
solvent in a stream of gas to form oil particles; directing light into the
stream of gas and the
oil particles, thereby forming reflected light: detecting the reflected light;
and, determining
the oil content based on the reflected light.
In a preferred embodiment. all fractions of oil of a sample are extracted. In
another
l0 preferred embodiment, triglycerides are used as a marker for the total oil
content. In this
embodiment, the signals produced by light scattering detection are derived
predominantly
from the triglyceride fraction of the total oil content.
The extracted oil can then be separated from solids in a centrifuge. To
determine oil
content, the supernatant can be injected into a device that is capable of
providing a stream of
gas in which the solvent can evaporate, and the mass of the remaining oil can
be determined
with an evaporative light scattering detector.
As used herein "oil content" refers to the amount of oil present in a sample
or
particular fraction or fractions of oil, e.g. 5 nanograms (ng) per seed of
total oil, 5 ng total oil
per 10 grams of dry weight of tissue. or 5 ng of triglycerides per seed, or 5
ng of triglycerides
2o per 10 grams of dry weight of tissue. Particularly preferred fractions of
oil include, without
limitation, triglycerides, free fatty acids, waxes, phospholipids,
phytosterols, and tocopherols.
In a preferred embodiment the fraction comprises triglycerides. As used
herein,
"composition" refers to biochemical constituents of an agricultural sample,
for example, the
ratio of triglycerides to total oil content. As used herein, "sample" means
any part of one or
z5 more plants being analyzed, including, for example, a portion of a seed. a
single seed, more
than one seed, a part of one or more plants other than seeds, any plant
tissue, agricultural
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material, or any combination thereof. A sample can be in any form, including
whole seeds,
intact plant tissue, whole agricultural material, and any disrupted form of
any of these.
Any seed can be utilized in a method of the present invention. In a preferred
embodiment, the seed is selected from the group consisting of alfalfa seed.
apple seed,
Arabidopsis thaliana seed, banana seed, barley seed, bean seed, broccoli seed.
castorbean
seed, citrus seed, clover seed, coconut seed, coffee seed, maize seed, cotton
seed, cucumber
seed, Douglas fir seed, Eucalyptus seed, Loblolly pine seed, linseed seed.
melon seed, oat
seed, olive seed, palm seed, pea seed, peanut seed, pepper seed, poplar seed.
Radiata pine
seed, rapeseed seed, rice seed, rye seed, sorghum seed, Southern pine seed.
soybean seed,
to strawberry seed, sugarbeet seed, sugarcane seed, sunflower seed, sweetgum
seed, tea seed,
tobacco seed, tomato seed, turf, and wheat seed. In a more preferred
embodiment, the seed is
selected from the group consisting of cotton seed, Arabidopsis thaliana seed.
maize seed,
soybean seed, rapeseed seed, rice seed, and wheat seed. In an even more
preferred
embodiment, the seed is a rapeseed seed. In another even more preferred
embodiment, the
15 seed is an Arabidopsis thaliana seed. In another even more preferred
embodiment, the seed
is a soybean seed. In yet another even more preferred embodiment, the seed is
a maize seed.
Further, any portion of any of the above-mentioned seeds can be utilized. For
example, any of the above-mentioned seeds can be subdivided for the purposes
of analysis. A
seed can, for example, be divided so as to bisect the germ and endosperm in
order to allow for
2o parallel testing and planting of the two halves. A seed can further be
divided by tissue type.
In a preferred embodiment, a sample can comprise endosperm that has been
mechanically
separated from the germ tissue in order to analyze the germ or the endosperm
for oil content
using a method of the present invention.
Other plant tissues or agricultural materials can be substituted, without
limitation, for
25 seeds as the sample. As used herein, plant tissues include, without
limitation, any plant part
such as leaf, flower, root, petal. As used herein, agricultural materials
include, without
limitation, plant tissues such as seeds. but also include, without limitation.
non-plant based
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material such as non-organic matter or non-plant based matter that occur in an
agricultural
context. Fungal samples are an example of an agricultural material.
Individual seeds or batches of seeds can be utilized with the methods of the
present
invention. A batch of seeds is any number of seeds greater than one. As used
herein, a
"member" of a batch is any single seed within the batch. A batch of seeds can
be defined by
number. In an embodiment, a batch of seeds is greater than 10,000, 5,000,
2,500, 1,000, 100,
20, 10, 5, 4, or 3 seeds. In another embodiment a batch comprises between
5,000 and 10,000
seeds, between 1,000 and 5,000 seeds, 100 and 2,500 seeds, 100 and 1,000
seeds, 10 and 100
seeds, 10 and 20 seeds, 5 and 10 seeds, 1 and 5 seeds, 1 and 4 seeds, and 1
and 3 seeds. In
to another embodiment the batch of seeds may be classified by its origin, such
as seeds that are
derived from a single ear, single plant, or single plant cross.
In one embodiment, the seeds from a single source are provided together for
analysis.
In another embodiment the single source can be any source that provides seeds
having a
similar genetic background, such as an ear of maize, a single plant, or the
product of a single
cross. If a seed or a batch of seeds is entirely consumed by a method of the
present invention,
then seeds having a common genetic background can be used to propagate a
desired trait
found in an analyzed seed.
As used herein, a seed with a similar genetic background to a first seed is a
seed that
shares at least 25%, more preferably 50%, even more preferably 75% or 100% of
the genetic
2o background of the first seed. For example, the progeny of a cross between
two plants shares
50% of its genetic background with each parent to the cross.
The mass of a sample can be any mass that yields a measurable result. In a
preferred
embodiment, the sample mass is less mass than 1,000 grams, more preferably
less than 500,
100, 50. 25, 10, 5, and 1 gram. In another preferred embodiment, the sample is
one seed.
In order to determine the oil content of a sample, the oil is extracted from
the sample.
Extraction can be performed on the sample using a suitable solvent. The
solvent can be any
solvent that is capable of extracting oil from the sample without also
extracting unwanted
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impurities from the sample. In a preferred embodiment, the solvent is any non-
polar solvent.
In a further preferred embodiment, the solvent is selected from the group
consisting of
hexane, decane, petroleum ether, an alcohol, or acetontirile. In a preferred
embodiment the
solvent comprises isopropanol. In a more preferred embodiment, the solvent
comprises
hexane.
The amount of solvent used will depend upon the amount of sample analyzed. The
volume of solvent sufficient to extract a detectable amount of oil is known in
the art. In a
preferred embodiment, sufficient solvent is used to extract all available oil
from the sample.
The available oil in a sample can be the total oil in the sample, or any
amount less than the
to total amount of oil. In another embodiment, 0.1 to 100 milliliters of
solvent is used for every
milligram of sample being analyzed, with 0.2 to 50 milliliters of solvent per
milligram of
sample preferred, 0.25 to 10 milliliters of solvent per milligram of sample
more preferred,
and 0.5 to 3 milliliters of solvent per milligram especially preferred.
As used herein, "extracting oil" from a sample means disposing the sample in
contact
with a solvent in order to transfer oil from the sample to the solvent. During
extraction, a
sample can be exposed to the solvent in any manner that transfers a detectable
amount of oil
from the sample to the solvent. A sample can, for example. be added to an
appropriate
volume of solvent in an intact state. Oil then is drawn from the intact sample
to the solvent.
In one embodiment, the oil is held in solution in the solvent.
2o In order to increase the rate of oil transfer or the amount of oil that is
transferred
during extraction, a sample can be disrupted. As used herein, "disrupting" a
sample means
physically altering a sample in order to increase the surface area of the
sample that can be
exposed to a solvent. Disrupting can be performed with anv suitable device,
including
devices for grinding, milling, crushing, cutting, and pulverizing, among
others. A Tecator
Cyclotec 1093 Sample Mill (Fos Tecator, P.O. Box 70, S-26321 Hoeganaes,
Sweden) is one
example of a commercially available milling device.
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In order to increase the rate of oil transfer or the amount of oil transferred
during
extraction, the combined sample and solvent mixture can be agitated. As used
herein,
"agitating" a solvent and sample means using any technique to increase the
physical
interaction of the solvent and sample. Agitation of the solvent and sample can
be performed,
for example, with vibrators, agitators, rotating wheels, and shakers, among
others. One
example of an agitation device is a Glas-Col rotating wheel (Glas-Col
Apparatus Co, 711
Hulman Street, P O Box 2128, Terre Haute, IN 47802-0128
USA). Additionally, the temperature of the solvent and the sample can be
increased in order
to improve the rate of oil transfer or the amount of oil transferred.
The amount of disrupting and agitating of a sample will depend on the desired
result
of the analysis. In some instances it will be desirable to compare the
relative oil content of
two or more samples. In these instances, it is unnecessary to extract the
total oil content from
the sample. Instead, a portion of the total oil content can be extracted from
each sample and
the amounts can be compared to determine the relative oil content of the
samples. In one
embodiment, in order to determine relative oil content, disruption and
agitation of samples
can be minimal. For example, multiple samples can be disposed in solvent
without disrupting
the sample beforehand, and then agitated. After centrifugation and evaporative
light
scattering detection, the relative oil content of the samples can be
ascertained.
In another embodiment, a quantitative estimation of total oil content is
obtained by
disrupting a sample and agitating the sample in solvent in order to transfer
almost all of the
oil from the sample to the solvent. The extent to which a sample will need to
be disrupted
and agitated in order to liberate all of the oil content depends on the type
of sample under
analysis and is known in the art.
In a preferred embodiment, a sample is ground for between 0.1 and 5 minutes in
a
sample mill, and more preferably between 0.5 and 3 minutes. In another
embodiment, a
sample is agitated for between 0.5 and 20 minutes, more preferably between 1
and 15
minutes. and even more preferably from 3 to 8 minutes.
l0
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After extraction, the solvent and extracted oil, which is in solution in the
solvent. can
be further separated from the remaining sample in order to improve the
uniformity of the
composition of the solvent and extracted oil. Any device and method for
separating solids
from a solution can be used if the resulting solvent comprises an amount of
non-oil impurities
that does not significantly affect detection of oil in later steps. One
example of a suitable
centrifuge is a Fisher Model 235B Micro-Centrifuge (Fisher Scientific, 4500
Turnberry Dr.,
Hanover Park, IL 60103). As used herein, "separating" solvent from seed means
removing
the solvent containing the extracted oil from the remainder of the sample.
Separation can be
performed, for example, with anv conventional technique, including filtration,
settling, and
1o centrifugation. In a preferred embodiment, after extraction the solvent and
sample solids are
centrifuged. Centrifugation causes the solids to sediment and form a pellet,
from which the
solvent is separated as supernatant. The supernatant can then be siphoned off
of the pellet to
complete the separation. In a preferred embodiment, the sample and solvent are
centrifuged
for between 0.1 to 5 minutes, more preferably for between 0.5 and 3 minutes,
and even more
preferably for between 0.75 and 2 minutes.
After extraction of the oil from the solvent, the oil content in the solvent
can be
determined using evaporative light scattering detection methods. Any device
that is capable
of providing a stream of gas in which the solvent can evaporate and form oil
particles in
solvent vapor can be used in conjunction with a light source capable of
producing light that is
2o reflected by the oil particles and a light detector capable of detecting
the reflected light.
As used herein, "evaporating solvent" means causing the solvent in the solvent
and oil
solution to go to a gaseous vapor phase from a liquid phase, while maintaining
the oil in a
liquid phase. Evaporation of the solvent results in free oil particles, or
droplets, that can then
be passed through a light source and light detector for determination of the
mass of the oil.
Evaporation of the solvent can be earned out in any device that is capable of
providing a
stream of gas in which the solvent can evaporate. As used herein, a "stream of
gas" is a
continuous flow of gas in which a solvent can be evaporated. The gas used in
the stream of
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gas can be any gas into which the solvent can evaporate. In one embodiment,
the gas is an
inert or noble gas. In another embodiment, the gas is selected from the group
consisting of
nitrogen, inert or noble gases, and carbon dioxide and mixtures thereof. In
another preferred
embodiment, the gas used in the stream of gas comprises nitrogen. In yet
another preferred
embodiment, the gas used comprises nitrogen of at least 99% purity.
The device that is capable of providing a stream of gas in which the solvent
can
evaporate can be any conventional device used for evaporative light scattering
detection. In
one embodiment, the device is a tube that is affixed at one end to a different
device for
supplying the solvent at a controllable rate. Such a tube can be a "drift"
tube as are known in
to the art. The tube can have any geometry that allows for the evaporation of
the solvent, with
an approximately cylindrical geometry preferred. At one end of the tube, a
device for
introducing the solvent at a controlled rate into the tube is provided.
The rate at which the stream of gas flows can be any rate that allows for
sufficient
evaporation of a solvent in a stream of gas. The rate at which the stream of
gas flows will
depend on which solvent and gas are used, the relative amounts of each, the
temperature of
each, and the time of contact of the two. In a preferred embodiment, the flow
rate of gas in
the tube is between 0.5 and 20 liters per minute, more preferably between l
and 10 liters per
minute, and even more preferably betvveen 1.5 and 5 liters per minute. In a
preferred
embodiment, the solvent is introduced into the stream at a rate between 0.1
and 10 milliliters
2o per minute, more preferably between 0.2 and 5 milliliters per minute, and
even more
preferably between 0.3 and 5 milliliters per minute. In a preferred embodiment
the
temperature of the solvent is between 20 and 100 degrees Celsius, more
preferably between
30 and 75 degrees Celsius, and even more preferably between 30 and 50 degrees
Celsius. In
a preferred embodiment the tube is maintained at a temperature between 20 and
200 degrees
Celsius, more preferably between 50 and 150 degrees Celsius, and even more
preferably
between 80 and 120 degrees Celsius.
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The solvent can be introduced into a device that is capable of providing a
stream of
gas in which the solvent can evaporate in any manner and with any device for
introducing the
solvent at a controlled rate. Devices that allow for the delivery of a
constant volume of
solvent over time include any device for introducing the solvent at a
controlled rate. For
example, the device can be a liquid pump with a pressure regulator. The device
for
introducing the solvent at a controlled rate can be connected to the device
that is capable of
providing a stream of gas in which the solvent can evaporate in any manner
that allows for
the distribution of the solvent. In one embodiment, the device that is capable
of providing a
stream of gas in which the solvent can evaporate comprises a nebulizer, which
is used to mix
io gas and solvent and inject the resulting mixture into the device in a
dispersed spray. In this
embodiment, the solvent is injected into the nebulizer by a device for
introducing the solvent
at a controlled rate, and the solvent is mixed with a steam of gas and
injected into the device
that is capable of providing a stream of gas in which the solvent can
evaporate. As the stream
of gas and solvent proceeds through the device, the solvent evaporates,
leaving dispersed
particles of oil in the stream of gas. The particles of oil then pass through
the light and form
reflected light, which is detected. In this embodiment, the solvent introduced
into the device
that is capable of providing a stream of gas in which the solvent can
evaporate will comprise
a generally uniform concentration of oil, and the resulting signals produced
by the light
detector will change from a zero reading, to a maximum reading, and then back
to a zero
2o reading over time.
In another embodiment, a continuous volume of a second solvent can be provided
to
the device that is capable of providing a stream of gas in which the solvent
can evaporate. In
this embodiment, the solvent with the oil content is introduced into the
second solvent while
the second solvent is being constantly provided. In this manner, the signals
produced by the
light detector will change in intensity more gradually, and can accurately be
charted, with the
oil content represented as a peak. All of the components described herein can
be used for the
various components in this embodiment.
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In addition, a conventional high-performance liquid chromatography (HPLC)
device
can be used as the device that can regulate the volume or pressure of a fluid.
In this
embodiment, in the mobile phase of the HPLC device is a second solvent, which
includes,
without limitation, any of the solvents mentioned herein, and which can be the
same as or
different than the solvent with extracted oil. In a preferred embodiment, the
second solvent
comprises isopropanol and hexane. In another preferred embodiment, the second
solvent
comprises 10% isopropanol and 90% hexane. Any HPLC device that is capable of
supplying solvent to the device that is capable of providing a stream of gas
in which the
solvent can evaporate can be used. Examples of suitable HPLC devices include a
Hewlett-
1o Packard 1090 with a Micra NPS, 33X4.6 millimeter, 1.5 micron plus Guard
Column. In this
embodiment, the second solvent can be provided continuously.
Before addition of the solvent with the extracted oil to the second solvent,
the light
detector will not detect any reflected light, because the second solvent
evaporates prior to
reaching the light. The solvent with the extracted oil is then added to the
flow of the second
solvent, and is carried into either the HPLC column or directly to the device
that is capable of
providing a stream of gas in which the solvent can evaporate. In either
embodiment, the
solvent with the extracted oil becomes dispersed in the second solvent prior
to reaching the
device that is capable of providing a stream of gas in which the solvent can
evaporate.
Upon introduction into the device that is capable of providing a stream of gas
in
2o which the solvent can evaporate, both solvents evaporate, and the oil is
carried toward the
light and light detector in the stream of gas. Since the solvent with the
extracted oil is
dispersed in the second solvent, the light detector will signal an increase
from the zero
baseline of the evaporated second solvent alone to a peak of oil content, and
then back to the
baseline of the evaporated second solvent when the oil particles have
completely passed the
light detector.
The HPLC is used as a readily available device for introducing the solvent at
a
controlled rate, and is not used here to separate components in the mobile
solvent phase. For
14
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embodiments incorporating a second, continuously supplied solvent, including
embodiments
using HPLC devices, preferred values for flow rates and temperatures include,
without
limitation, those described herein for embodiments using only one solvent. In
a preferred
embodiment, the volume of a solvent with extracted oil that is added to the
continuous flow
of the second solvent is between 0.5 and 50 microliters, more preferably
between 1 and 25
microliters, and even more preferably between 1 and 10 microliters. Although
the entire
sample of solvent with extracted oil can be added to the second solvent, any
portion of the
solvent with extracted oil can be added to yield a result that is proportional
to the fraction of
the solvent with extracted oil that was originally added.
The oil content of the solvent is determined with a light and a light
detector. The light
can be any light that is capable of being reflected by oil particles. Any
light source that
produces such light can be used as the light source. Potential light sources
include lasers and
collimated lasers. In a preferred embodiment, the light source is a 7
milliwattt, 670
nanometer laser diode. The light source is disposed so as to direct light into
the stream of
gas. As used herein, "directing light into a stream of gas" means providing a
light source so
that light emanating from the light source travels into the stream of gas. As
particles of oil
pass through the light, the light is reflected. As used herein, "reflected
light" is any light that
strikes an oil particle and is redirected toward the light detector.
The light detector can be any device that is capable of detecting light and
outputting a
2o signal that can be associated with the amount of light detected. In a
preferred embodiment
the light detected is reflected light. As used herein, "detecting reflected
light" means
generating a signal in response to light that has been produced by the light
source, has been
reflected by the extracted oil, and has struck the light detector. Suitable
light detectors
include, without limitation, silicon photodiodes, photomultipliers, and photon
counters. In a
preferred embodiment, a photodetector, which is a silicon photodiode, is used
as the light
detector. The photodetector in this embodiment outputs a signal that is
proportional to the
amount of reflected light striking the detector. The photodetector is
preferably disposed at an
CA 02361679 2001-07-24
WO 01/23884 PCT/US00/26374
angle relative to the path of the light. In one embodiment, the angle between
the line of the
light and the light detector is between 45 degrees and 135 degrees, with the
vertex of the
angle in the center of the long axis of the stream of gas and each line in the
angle lying in the
same plane, which is perpendicular to the long axis of the stream of gas. The
light source and
the light detector can be disposed at any distance from the stream of gas that
allows for
sufficient formation of reflected light and sufficient detection of that
reflected light.
In order to associate the signals from a light detector with a quantity of
oil,
calibrations are performed with the methods of the present invention using a
known amount
of oil in a solvent. As used herein, "determining the oil content based on the
reflected light"
to means using known calibration values of oil content and reflected light to
determine oil
content. Any volume of oil can be used to calibrate a device described herein,
and in a
preferred embodiment, an oil volume approximating the predicted oil content of
a sample is
used for calibration. Light detector signals for known oil concentrations, for
example 0.1 to
3.5 milligrams per milliliter, can be used to create a model for correlating
actual oil levels
with light detector signals produced by a sample with unknown oil content.
The signals produced by the light detector can be sent to a device that is
capable of
storing or displaying the signal data. In one embodiment, a chromatograph is
used to plot the
amount of reflected light over time. In alternative embodiments, the signal
data can be sent to
data storage devices. computers, or a monitor. Once a plot of reflected light
over time is
2o produced, standard calculations can be used to determine the oil content as
represented by the
area under a peak on the curve of the plot.
The methods of the present invention are ideally suited to use in high
throughput
screening of seeds for oil content. The fast extraction time of the methods
are suitable for an
automated system using microtiter plates. In a preferred embodiment, the total
time for
2, extraction and determination of oil content of a sample is less than 10
minutes, more
preferably less than 8 minutes, and even more preferably less than 6.5
minutes. In another
preferred embodiment, relative oil content of samples is determined and
extraction and
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determination of oil content is done in less than five minutes, preferably
less than 3 minutes,
and even more preferably in less than 1.5 minutes.
Methods of the present invention can be used in a breeding program to select
plants or
seeds having a desired trait. In one aspect, the present invention provides a
method for
selecting a seed having an enhanced oil content. comprising: extracting oil
from a seed using
a solvent; evaporating the solvent in a stream of gas to form oil particles;
directing light into
the stream of gas and the oil particles, thereby forming reflected light;
detecting the reflected
light; determining the oil content based on the reflected light; and,
selecting a seed with a
similar genetic background to the seed based on the oil content. In another
aspect the present
1o invention provides a method of introgressing a trait into a plant
comprising: extracting oil
from a seed using a solvent; evaporating the solvent in a stream of gas to
form oil particles;
directing light into the stream of gas and the oil particles, thereby forming
reflected light;
detecting the reflected light; determining the oil content based on the
reflected light; selecting
a seed with a similar genetic background to the seed based on the oil content;
growing a
fertile plant from the related seed; and, utilizing the fertile plant as
either a female parent or a
male parent in a cross with a second plant.
The methods of introgression and selection of the present invention can be
used in
combination with any breeding methodology, and can be used to select a single
generation or
to select multiple generations. The choice of breeding method depends on the
mode of plant
2o reproduction, the heritability of the traits) being improved, and the type
of cultivar used
commercially (e.g., F, hybrid cultivar, pureline cultivar, etc). Selected, non-
limiting
approaches for breeding the plants of the present invention are set forth
below. A breeding
program can be enhanced using marker assisted selection of the progeny of any
cross. It is
further understood that anv commercial and non-commercial cultivars can be
utilized in a
breeding program. Factors such as, for example, emergence vigor, vegetative
vigor, stress
tolerance, disease resistance, branching, flowering, seed set, seed size, seed
density,
standability, and threshability etc. will generally dictate the choice.
17
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For highly heritable traits, a choice of superior individual plants evaluated
at a single
location will be effective, whereas for traits with low heritability,
selection should be based
on mean values obtained from replicated evaluations of families of related
plants. Popular
selection methods commonly include pedigree selection. modified pedigree
selection, mass
selection, and recurrent selection. In a preferred embodiment a backcross or
recurrent
breeding program is undertaken.
The complexity of inheritance influences choice of the breeding method.
Backcross
breeding can be used to transfer one or a few favorable genes for a highly
heritable trait into a
desirable cultivar. This approach has been used extensively for breeding
disease-resistant
to cultivars. Various recurrent selection techniques are used to improve
quantitatively inherited
traits controlled by numerous genes. The use of recurrent selection in self
pollinating crops
depends on the ease of pollination, the frequency of successful hybrids from
each pollination,
and the number of hybrid offspring from each successful cross.
Breeding lines can be tested and compared to appropriate standards in
environments
15 representative of the commercial target areas) for two or more generations.
The best lines
are candidates for new commercial cultivars; those still deficient in traits
may be used as
parents to produce new populations for further selection.
One method of identifying a superior plant is to observe its performance
relative to
other experimental plants and to a widely grown standard cultivar. If a single
observation is
20 inconclusive, replicated observations can provide a better estimate of its
genetic worth. A
breeder can select and cross two or more parental lines. followed by repeated
selfing and
selection, producing many new genetic combinations.
The development of new soybean cultivars entails the development and selection
of
soybean varieties, the crossing of these varieties and selection of superior
hybrid crosses. The
25 hybrid seed can be produced by manual crosses between selected male-fertile
parents or by
using male sterility systems. Hybrids are selected for certain single gene
traits such as pod
color, flower color, seed yield, pubescence color or herbicide resistance
which indicate that
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WO 01/23884 PCT/US00/26374
the seed is truly a hybrid. Additional data on parental lines, as well as the
phenotype of the
hybrid, influence the breeder's decision whether to continue with the specific
hybrid cross.
Pedigree breeding and recurrent selection breeding methods can be used to
develop
cultivars from breeding populations. Breeding programs combine desirable
traits from two or
more cultivars or various broad-based sources into breeding pools from which
cultivars are
developed by selfing and selection of desired phenotypes. New cultivars can be
evaluated to
determine which have commercial potential.
Pedigree breeding is used commonly for the improvement of self pollinating
crops.
Two parents who possess favorable. complementary traits are crossed to produce
an F,. An
to F, population is produced by selfing one or several F,'s. Selection of the
best individuals in
the best families is selected. Replicated testing of families can begin in the
F4 generation to
improve the effectiveness of selection for traits with low heritability. At an
advanced stage of
inbreeding (i.e., F6 and F,), the best lines or mixtures of phenotypically
similar lines are tested
for potential release as new cultivars.
Backcross breeding has been used to transfer genes for a simply inherited,
highly
heritable trait into a desirable homozygous cultivar or inbred line, which is
the recurrent
parent. The source of the trait to be transferred is called the donor parent.
The resulting plant
is expected to have the attributes of the recurrent parent (e.g., cultivar)
and the desirable trait
transferred from the donor parent. After the initial cross, individuals
possessing the
2o phenotype of the donor parent are selected and repeatedly crossed
(backcrossed) to the
recurrent parent. The resulting parent is expected to have the attributes of
the recurrent parent
(e.g., cultivar) and the desirable trait transferred from the donor parent.
The single-seed descent procedure refers to planting a segregating population,
harvesting a sample of one seed per plant, and using the one-seed sample to
plant the next
generation. When the population has been advanced from the F= to the desired
level of
inbreeding, the plants from which lines are derived will each trace to
different F, individuals.
The number of plants in a population declines each generation due to failure
of some seeds to
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WO 01/23884 PCT/US00/26374
germinate or some plants to produce at least one seed. As a result, not all of
the F= plants
originally sampled in the population will be represented by a progeny when
generation
advance is completed.
In a multiple-seed procedure, soybean breeders commonly harvest one or more
pods
from each plant in a population and thresh them together to form a bulk. Part
of the bulk is
used to plant the next generation and part is put in resen~e. The procedure
has been referred
to as modified single-seed descent or the pod-bulk technique.
The multiple-seed procedure has been used to save labor at harvest. It is
considerably
faster to thresh pods with a machine than to remove one seed from each by hand
for the
to single-seed procedure. The multiple-seed procedure also makes it possible
to plant the same
number of seed of a population each generation of inbreeding.
Descriptions of other breeding methods that are commonly used for different
traits
and crops can be found in one of several reference books (e.g. Fehr,
Principles of Cultivar
Development Vol. 1, pp. 2-3 (1987)), the entirety of which is herein
incorporated by
reference).
Figure 1 provides one embodiment of a system that is capable of performing the
methods of the present invention, which is shown generally at 10. A device for
introducing
the solvent at a controlled rate 12 is coupled with a source of a second
solvent 14. The device
12 pumps the second solvent at a controlled rate to a device that is capable
of providing a
2o stream of gas 16 in which the solvent can evaporate, which in this case
comprises a nebulizer
18 and a heated drift tube 20. A gas supply 22 is coupled to the nebulizer 18.
The second
solvent and the gas are mixed in the nebulizer 18, which causes the formation
of a disperse
stream of solvent droplets 24 in the drift tub 20. A light source 26 and a
light detector 28 are
disposed at an angle 0 in a plane that is perpendicular to the long axis 30 of
the drift tube 20.
In this example, 8 is 90 degrees. The light detector 28 outputs a signal 32
that is proportional
to the amount of reflected light striking the detector 28.
CA 02361679 2001-07-24
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To allow the introduction of the solvent with the extracted oil into the flow
of the
second solvent, a port 34 can be placed within the input line 36, or within
the device 12.
Finally, the light detector 28 can be coupled to a chromatograph 38 in order
to graphically
display the results of analyses.
Example 1
The detector is calibrated with known soybean oil concentrations of 0.1 to 3.5
milligrams of oil per milliliter of solvent. The results of this calibration
are shown in Figure
2. Figure 2 is a plot of area under a peak in a chromatograph (Y-Axis) versus
the amount of
extracted oil in the solvent (X-Axis). A sample of soybean is ground using a
Tecator
to Cyclotec 1093 Sample Mill (Fos Tecator, P.O. Box 70, S-26321 Hoeganaes,
Sweden).
Twenty milligrams of ground seed is added to a microcentrifuge tube. One
milliliter of
hexane is added to the tube, and the tube is agitated for five minutes on a
rotating wheel at
room temperature. The tube is then centrifuged in a microcentrifuge for five
minutes. The
supernatant is transferred to an HPLC autosampler vial and injected into an
HPLC apparatus.
Five microliters of hexane with extracted oil is added. The HPLC apparatus is
a Hewlett-
Packard 1090 with a Micra NPS, 33X4.6 millimeter, 1.5 micron plus Guard
Column. The
second solvent is 10%/90% isopranol/hexane, the flow rate is 0.5 milliliters
per minute, the
column temperature is 40 degrees Celsius, and the run time is 0.8 minutes. The
solvent is
nebulized in a Varex Evaporative light scattering detector with a drift tube
at 115 degrees
2o Celsius, an exit temperature of 55 degrees Celsius, and an attenuation of
1. The
chromatograph produced by this procedure is shown in Figure 3. In Figure 3,
the Y-axis
represents milliabsorbance units, which are units of reflected light intensity
that are
proportional to the mass of oil passing by the light detector. The X-axis
represents time, in
minutes. As can be seen in Figure 3. a base line of reflected light is
established from time
zero. From time zero until 0.5 minutes into the run, only the second solvent
is passing by the
light detector. At just after 0.5 minutes, the extracted oil begins to pass by
the light detector.
The extracted oil entirely passes the light detector by the 0.8 minute mark of
the run. The
21
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area under the peak centered at 0.594 is proportional to the mass of oil that
passed by the light
detector. The computed area under the peak curve can be located on the
calibration graph
shown in Figure 2 in order to determine the mass of the oil that passed by the
light detector.
Example 2
Figures 4-6 show the results of comparisons between the oil content of the
present
invention (light scattering, or LS) with the conventional technique of
accelerated solvent
extraction (ASE). The conditions for determination of oil content for LS are
as in example 1,
and ASE is performed using conventional practices. Figure 4 shows the percent
oil content as
determined for both LS and ASE, as well as the ratio (LS/ASE) of the oil
content as
to determined by each method for 12 different samples. As shown in Figure 4.
the ratios of oil
content fall significantly between 0.80 and 0.90. Figure 5 shows the same
ratio of oil content
for ten sorghum samples. Again, the ratios of oil content fall within a narrow
range,
significantly between about 0.60 and 0.65. Figure 6 shows the same ratio of
oil content for
maize. In this case, the ratios of oil content fall significantly between 0.7
and 0.8.
Example 3
Figure 7 demonstrates the reproducibility of one embodiment of the method of
the
present invention. As shown in Figure 7, the range of oil content determined
for ten identical
runs is between about 2.75 and 3.25 percent oil for the ten samples of maize.
The oil content
of the samples are determined as in example 1.
2p Example 4
Figure 8 is an overlayed chromatogram of three separate runs. The Y-axis and X-
axis
are milliabsorbance units and time, respectively. The three peaks shown
represent the peaks
produced by runs of one, two and three Arabodopsis thaliana seeds. Each of the
three
samples is extracted using 250 microliters of hexane. The second solvent is a
5/95 mixture of
Isopentanol/hexane, and the solvent is dispensed into the drift tube at a rate
of 1.0 milliliters
per minute. Conditions are otherwise as above for example two. The three
overlayed peaks
show the exceptional sensitivity and lower detection level of the methods of
the present
22
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WO 01/23884 PCT/US00/26374
invention. As seen in the plot, for each Arabidopsis thaliana seed included.
the area under
the peak increases equivalently.
The above-described invention provides methods of determining oil content of
samples with a higher sensitivity and lower detection limit than conventional
methods, and
that are amenable to automation. The methods can be rapidly performed with
readily
available devices on a wide variety of seeds, plant tissue, and agricultural
materials. Further,
unlike ultraviolet detection techniques, the methods of the present invention
are mass
sensitive, which allows for absolute quantition of oil content.
The above description, drawings and examples are only illustrative of
embodiments
1o which achieve the objects, features and advantages of the present
invention. It is not intended
that the present invention be limited to the illustrative embodiments.
23
