Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/47—Scattering, i.e. diffuse reflection
  • G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
  • G01N21/53—Scattering, i.e. diffuse reflection within a body or fluid within a flowing fluid, e.g. smoke
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/02—Food
  • G01N33/03—Edible oils or edible fats
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/47—Scattering, i.e. diffuse reflection
  • G01N2021/4704—Angular selective
  • G01N2021/4709—Backscatter
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/47—Scattering, i.e. diffuse reflection
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/62—Detectors specially adapted therefor
  • G01N30/74—Optical detectors

Definitions

• 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 more seeds.
• NIR near infrared analysis
• NMR nuclear magnetic resonance imaging
• ASE accelerated solvent extraction
• microwave extraction microwave extraction
• super critical fluid extraction are often not able to accurately discern the relative or absolute levels of oil in very small seed samples.
• NIR near infrared
• this non-destructive technique is also often not suitable for the analysis of seed oil when the seed of interest is small.
• 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 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.
• 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.
• 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 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 ag ⁇ cultural mate ⁇ al, composing: extracting oil from the mate ⁇ al 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, composing: extracting oil from the batch seed sample 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 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 mtrogressing 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 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) through e) one or more times, and, g) selecting one or more seeds based on the oil content.
• 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.
• 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 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.
• Figure 8 is a overlayed chromatograph showing oil content determination for one, two, and three Arabidopsis thaliana seeds. Detailed Description Of The Invention
• the present invention provides analytical methods for selecting seeds having a desired oil content.
• 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.
• all fractions of oil of a sample are extracted.
• triglycerides are used as a marker for the total oil content.
• 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.
• 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.
• 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 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.
• the fraction comprises triglycerides.
• composition refers to biochemical constituents of an agricultural sample, for example, the ratio of triglycerides to total oil content.
• sample means any part of one or 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 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.
• 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.
• 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.
• the seed is a rapeseed seed.
• the seed is an Arabidopsis thaliana seed.
• the seed is a soybean seed.
• the seed is a maize seed.
• any portion of any of the above-mentioned seeds can be utilized.
• 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 parallel testing and planting of the two halves.
• a seed can further be divided by tissue type.
• 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.
• plant tissues include, without limitation, any plant part such as leaf, flower, root, petal.
• agricultural materials include, without limitation, plant tissues such as seeds, but also include, without limitation, non-plant based 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.
• a batch of seeds is any number of seeds greater than one.
• a "member" of a batch is any single seed within the batch.
• a batch of seeds can be defined by number.
• a batch of seeds is greater than 10,000, 5,000, 2,500, 1,000, 100, 20, 10, 5, 4, or 3 seeds.
• 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.
• 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.
• the seeds from a single source are provided together for analysis.
• 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.
• 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 background of the first seed.
• 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.
• the sample mass is less mass than 1 ,000 grams, more preferably less than 500, 100, 50. 25, 10, 5, and 1 gram.
• the sample is one seed.
• the oil is extracted from the sample.
• the solvent can be any solvent that is capable of extracting oil from the sample without also extracting unwanted impurities from the sample.
• the solvent is any non-polar solvent.
• the solvent is selected from the group consisting of hexane, decane, petroleum ether, an alcohol, or acetontirile.
• the solvent comprises isopropanol.
• 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.
• 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 total amount of oil.
• 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.
• 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.
• 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.
• a sample can be disrupted.
• "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 any 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.
• the combined sample and solvent mixture can be agitated.
• "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 Hul an Street, P O Box 2128, Terre Haute, IN 47802-0128
• 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.
• 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.
• a sample is ground for between 0.1 and 5 minutes in a sample mill, and more preferably between 0.5 and 3 minutes.
• 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.
• 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.
• 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 any conventional technique, including filtration, settling, and centrifugation.
• 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.
• 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.
• 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 reflected by the oil particles and a light detector capable of detecting the reflected light.
• 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 carried out in any device that is capable of providing a stream of gas in which the solvent can evaporate.
• a "stream of gas” is a continuous flow of gas in which a solvent can be evaporated.
• the gas used in the stream of gas can be any gas into which the solvent can evaporate. In one embodiment, the gas is an inert or noble gas.
• the gas is selected from the group consisting of nitrogen, inert or noble gases, and carbon dioxide and mixtures thereof.
• the gas used in the stream of gas comprises nitrogen.
• 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.
• the device is a tube that is affixed at one end to a different device for supplying the solvent at a controllable rate.
• a tube can be a "drift" tube as are known in the art.
• the tube can have any geometry that allows for the evaporation of the solvent, with an approximately cylindrical geometry preferred.
• a device for introducing the solvent at a controlled rate into the tube is provided at one end of the tube.
• 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.
• the flow rate of gas in the tube is between 0.5 and 20 liters per minute, more preferably between 1 and 10 liters per minute, and even more preferably between 1.5 and 5 liters per minute.
• the solvent is introduced into the stream at a rate between 0.1 and 10 milliliters per minute, more preferably between 0.2 and 5 milliliters per minute, and even more preferably between 0.3 and 5 milliliters per minute.
• 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.
• 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.
• 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.
• 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.
• 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 gas and solvent and inject the resulting mixture into the device in a dispersed spray.
• 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.
• 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.
• 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 reading over time.
• 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.
• the solvent with the oil content is introduced into the second solvent while the second solvent is being constantly provided.
• 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.
• a conventional high-performance liquid chromatography (HPLC) device can be used as the device that can regulate the volume or pressure of a fluid.
• a second solvent 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.
• the second solvent comprises isopropanol and hexane.
• 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- Packard 1090 with a Micra NFS, 33X4.6 millimeter, 1.5 micron plus Guard Column.
• the second solvent can be provided continuously.
• the light detector 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.
• 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.
• preferred values for flow rates and temperatures include, without limitation, those described herein for embodiments using only one solvent.
• 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.
• 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 signal that can be associated with the amount of light detected.
• the light detected is reflected light.
• 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.
• 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 angle relative to the path of the light.
• 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.
• determining the oil content based on the reflected light 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.
• a chromatograph is used to plot the amount of reflected light over time.
• the signal data can be sent to data storage devices, computers, or a monitor. Once a plot of reflected light over time is 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.
• the total time for 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.
• relative oil content of samples is determined and extraction and 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.
• 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.
• the present 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 reproduction, the heritability of the trait(s) 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 any 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.
• a choice of supe ⁇ or individual plants evaluated at a single location will be effective, whereas for traits with low he ⁇ tabihty, 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
• Breeding lines can be tested and compared to approp ⁇ ate standards environments representative of the commercial target area(s) 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 supe ⁇ or plant is to observe its performance relative to other expe ⁇ mental plants and to a widely grown standard cultivar If a single observation is 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 va ⁇ eties, the crossing of these va ⁇ eties and selection of supe ⁇ or hyb ⁇ d crosses
• the hyb ⁇ d seed can be produced by manual crosses between selected male-fertile parents or by using male ste ⁇ hty systems
• Hyb ⁇ ds are selected for certain single gene traits such as pod color, flower color, seed yield, pubescence color or herbicide resistance which indicate that 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 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 F 4 generation to improve the effectiveness of selection for traits with low heritability. At an advanced stage of inbreeding (i.e., F 6 and F 7 ), 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.
• individuals possessing the 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.
• 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 germinate or some plants to produce at least one seed. As a result, not all of the F 2 plants originally sampled in the population will be represented by a progeny when generation advance is completed.
• 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 reserve.
• 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 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.
• 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 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 ⁇ in a plane that is perpendicular to the long axis 30 of the drift tube 20. In this example, ⁇ is 90 degrees.
• the light detector 28 outputs a signal 32 that is proportional to the amount of reflected light striking the detector 28.
• a port 34 can be placed within the input line 36, or within the device 12.
• the light detector 28 can be coupled to a chromatograph 38 in order to graphically display the results of analyses.
• FIG. 1 is a plot of area under a peak m a chromatograph (Y-Axis) versus the amount of extracted oil m the solvent (X-Axis).
• Y-Axis a chromatograph
• X-Axis a chromatograph
• a sample of soybean is ground using a Tecator 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 m a Varex Evaporative light scatte ⁇ ng detector with a d ⁇ ft tube at 115 degrees Celsius, an exit temperature of 55 degrees Celsius, and an attenuation of 1.
• the chromatograph produced by this procedure is shown m 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.
• 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 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 m order to determine the mass of the oil that passed by the light detector
• Example 2 Figures 4-6 show the results of compa ⁇ sons between the oil content of the present invention (light scatte ⁇ ng, 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 determined by each method for 12 different samples
• 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
• 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
• Figure 7 demonstrates the reproducibihty of one embodiment of the method of the present invention
• 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 m example 1
• Figure 8 is an overlayed chromatogram of three separate runs
• the Y-axis and X-axis are mil absorbance 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 d ⁇ ft 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 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.
• the methods of the present invention are mass sensitive, which allows for absolute quantition of oil content.

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