CN101578509A - Clip based sampling of seed for the removal of specific seed tissue or structures for seed analysis - Google Patents

Abstract

The present invention discloses devices, methods and systems for useful seed sampling wherein viability is maintained. Seeds from one generation in a plant evolution experiment were individually sampled by removing and collecting the tissue of the seeds using a hand-held and hand-operated tool with one or more blades. The tissue is then processed in order to obtain one or more biochemical, genetic, or dominant characteristics of the seed, after which a decision is made whether to use the seed in further plant evolution experiments or in other plant research and development decision. In some embodiments of the methods, sampling is controlled so as to remove useful amounts of tissue for analytical purposes without significantly affecting the likelihood of viability of the sampled seeds. In some embodiments, sampling is controlled to prevent sample contamination. In certain embodiments, the seeds are held in a predetermined orientation to facilitate efficient and accurate sampling.

Description

Pruning-based seed sampling to remove specific seed tissues or structures for seed analysis

【Technical field】

The present invention relates to an efficient method for obtaining tissue samples from individual seeds.

【Background technique】

It is common practice in plant breeding or plant evolution experiments to grow plants from seeds of known origin. Seeds are planted in test plots, growth chambers, greenhouses, or other growing environments where they are either cross-pollinated with plants of other known origin or self-pollinated. The seeds produced are progeny from both parent plants or self-pollinated plants, and the seeds are harvested, processed and planted to continue the plant breeding cycle. To aid in the cultivation or to facilitate the selection process, specific laboratory or field-based tests may be performed on the plants, plant tissue, seeds or seed tissue.

Plant generations based on known crosses or self-pollination are grown and then tested to see if the lines or varieties are moving towards the characteristics desired in the marketplace. Examples of desirable traits include, but are not limited to, increased yield, improved purity, newly conferred resistance and/or tolerance or improvement thereof to specific herbicides and/or pests and pathogens, increased oil content, Modifications in starch content, nutraceutical composition, drought tolerance and enhancement of specific morphological-based traits.

As can be appreciated and known in the art, the scale of these experiments can be enormous. They require a vast workforce, from scientists to field workers, to design, grow, maintain and conduct experiments that can involve thousands of individual plants. They also require large land resources. Fields or greenhouses can occupy thousands of acres of land. As plants germinate, grow and produce seeds, this takes up a lot of land for months, during which time the seeds may not only be sampled for laboratory or field testing, but then the large number of seeds must be individually labeled, harvested and processed.

Another difficulty was that many experiments were fruitless. It has been reported in the literature that some seed companies discard 80-90% of the plants of any generation early in the experiment. Therefore, most of the large amount of land, manpower and material resources spent on growing, harvesting and post-harvest processing of seeds ends up being wasted.

Time pressure is also a factor. Major advances in plant breeding will put more pressure on seed companies to more rapidly advance lines or varieties of plants to achieve more and better traits and characteristics. Thus, plant breeders and related workers are under increased pressure to process these plants more efficiently and vigorously, and to make more and earlier selections for plants that should continue into the next generation of breeding

Thus, there has been a movement to identify traits of interest at an early stage through laboratory-based seed testing. Non-destructive testing of seeds to derive genetic, biochemical or phenotypic information. If the trait of interest is identified, then selected seeds from a particular plant are used for further experimentation and evolution or for the production of commercial quantities. Testing the seeds avoids the need to grow the seeds into immature plants, which are then tested. This saves time, space and effort. Effective identification of desired traits in seeds at an early stage can lead to greatly reducing the amount of land available for experimental testing, reducing the amount of seeds that must be tested and reducing the amount of time required to derive the information needed for evolutionary testing. For example, instead of thousands of acres of plants and subsequent processing and handling of all those plants, a small fraction of the land and plants will suffice. However, since timing is still important, since even this reduction involves processing eg thousands of seeds per day, this is still an essential task.

A traditional approach to seed sampling that attempts to be non-lethal is given below. Use tweezers to immobilize individual seeds of interest on a piece of paper spread out on a surface. Small drill bits are used to drill into small spots on the seeds. Debris removed from the seeds by the drill was collected on the paper. The paper is picked up and the pieces are transported to a test tube or other container. It is thus collected and ready for laboratory analysis. This method is intended not to destroy the seed. However the processing is slower. Its success and effectiveness depend heavily on the attention and precision of its staff. Each seed must be picked and fixed by hand with tweezers. Drilling is likewise done by hand. Drilling and machining of chips must be done with care. A single container (eg, a single test tube) must then be processed and labeled, or otherwise tracked and identified. Additionally, the tweezers and drill holes must be clean between the various sub-samples. There can be significant contamination risks through sample-to-sample carryover and manual handling. Moreover, it is often desired to obtain seed material from a certain physiological tissue of the seed. For example, it may be desirable to obtain a sample from the endosperm of corn seeds. In this case, it is not only tedious but time consuming, and there is some difficulty in manually grasping the small corn seeds in this manner, allowing the endosperm to be oriented so that it is exposed for drilling. Sampling from other seed structures such as the seed germ must be avoided because sampling from such seed regions has a negative impact on germination rates. Sometimes it is difficult to obtain a useful amount of sample using this method. In summary, sampling from seeds is heavily dependent on worker skill and is related to throughput and accuracy, including whether the program gives seeds a good chance to germinate. These problems are magnified when workers are tasked with processing many seeds in a day.

Serial Extraction of Endosperm Drillings published by V.Sangtong, E.C.Mottel, M.J.Long, M.Lee and M.P.Scott in June 2001 in Plant Molecular Biology Reporter 19:151-158 (Plant Molecular Biology Report: 19:151-158) (SEED)-Method for Detecting Transgenes and Proteins in Single Viable Maize Kernels ("Serial Extraction of Seed Endosperm Drills"-a method for detecting transgenes and proteins in single viable maize kernels) discloses the extraction of transgenes and proteins from maize seeds Another example of non-lethally obtaining a tissue sample for laboratory analysis, which is hereby incorporated by reference. This example describes the use of a hand-held rotary mill to grind away particles from the nucleus (so-called "borer") and the collection of particles for testing for the presence of a certain gene. Particles are directed into multi-well trays or individual wells of plates for identification and preparation for subsequent assays and analysis. However, this method also requires manual grasping and orientation of each seed relative to the grinder. This is also time consuming and somewhat cumbersome. It also relies on the skills of the staff. The method increases throughput and precision, availability of useful sample amounts and contamination results. To avoid contamination, the grinder must be thoroughly cleaned between each sample.

As demonstrated by these examples, existing traditional methods of seed analysis, such as those used in genetic, biochemical or phenotypic analysis, require the removal and manipulation of at least a portion of the seed. In removing some seed tissue, various goals may need to be met. These goals can include one or more of the following:

(a) Maintain the viability of the sampled seeds.

(b) Obtain at least the minimum necessary sample size without compromising viability.

(c) Obtaining a sample from a specific location on the seed usually requires the ability to orient the seed at a specific location for sampling.

(d) Maintain a specific throughput level for efficiency purposes.

(e) Reduce or virtually eliminate contamination between samples.

(f) Allows tracking of different samples and correlations to other samples in the set.

(a) Viability

With regard to maintaining seed viability, it may be critical in situations where seed sampling methods and devices do not damage the seed in such a way that seed viability is reduced. It is generally desirable that such an analysis be non-lethal to the seed, or at least result in a basic probability that the sampled seed will germinate (eg, without significantly reducing germination potential), so that the seed can grow into a mature plant. For some analyses, seed viability does not need to be maintained, in which case larger samples can usually be used. The need for seed viability will depend on the intended use of the seed after sampling.

(b) Sample size

It is desirable to obtain a useful amount of sample. In some applications it is useful that the sample size must be higher than some minimum necessary in order to perform a given test and obtain meaningful results. Different tests or analyzes require different sample sizes. It may be equally important to avoid obtaining too much tissue for a sample, as too large a sample can reduce the germination potential of the seeds, which may be undesirable. Accordingly, it is desirable that sampling devices and methods allow for variation in the amount of sample obtained from any given seed.

(c) Sample location

A useful amount of samples can also include sample location accuracy. For example, in some applications, samples must only come from a certain location or a certain tissue. Furthermore, it is difficult to process small particles like many seeds. It is also difficult to precisely locate and orient the seeds. For example, with corn seeds, it may be important to extract germ tissue and to orient the corn seeds so that specific tissues are extracted. Accordingly, it is desirable that sampling devices and methods be adapted to allow sampling of specific locations, which may include specific seed orientation methods.

(d) Throughput

Sampling devices and methods must allow for a throughput level that supports obtaining the required number of samples in a time efficient manner. For example, some situations involve potential demands for hundreds, thousands, or even millions of seeds per year. Assuming an example of sampling millions of seeds per year and a 5-day week, this would average close to four thousand samples per day for each weekday of the year. This need is difficult to meet using lower throughput sample methods. Therefore, a higher throughput, automatic or even semi-automatic approach may be desired.

(e) Avoid pollution

In order to maintain the purity of samples used in subsequent analytical testing procedures, it is desirable that sampling methods and devices are not prone to cross-contamination. This can include not only sample positioning accuracy so that a sample from a specific location is not contaminated by tissue from a different location, but also the sampling method and processing of each individual sample to ensure no contamination between samples.

(f) Tracking samples

Efficient handling of seeds and samples removed from seeds presents various problems and challenges, not least of which is keeping track of individual seeds, individual samples and their relationship to each other or to other samples in a batch. Accordingly, it is desirable that sampling devices and methods allow for easy tracking of seeds and samples.

Traditional seed sampling techniques do not adequately address these needs, resulting in material and human strain, and thus showing a need for improving the state of the art. Due to the reliance on significant manual processing, orientation, and removal of samples from seeds, current methods have relatively low throughput, carry substantial risk of cross-contamination, and tend to be inconsistent. This can affect the type of sample taken from the seed and the likelihood that the seed will germinate. There is a need to eliminate the resources that current methods require for inter-sample cleaning. There is a need to reduce or minimize cross-contamination between samples due to carryover or otherwise, or any contamination from any source of any sample. There is also a need for more reliability and precision. Therefore, there is a need for methods and their corresponding devices that provide for seed sampling, thereby achieving one or more of the following objectives:

(a) maintain seed viability after sampling;

(b) obtain at least the minimum necessary sample size without compromising viability;

(c) obtaining a sample from a specific location on the seed;

(d) maintaining a specified throughput level for efficiency purposes;

(e) reduce or virtually eliminate contamination between samples; and

(f) Allows tracking of different samples and their interrelationships with other samples in the set.

Some of these purposes are desired as sampling seeds may conflict and even contradict. For example, obtaining a useful sample volume while maintaining seed viability requires obtaining some seed tissue, but not too much. High-throughput methods may require relatively fast runs with relatively high precision and low risk of contamination, so they must be slower than technically possible. Accordingly, these multiple objectives exist in the prior art and have not been satisfactorily addressed and balanced by existing suitable methods and devices.

As will be appreciated, semi-automation or full automation of processing can generally satisfy at least some of the objectives (a)-(f) listed above. However, automation also involves basic cost, complexity and organization. It is not portable and takes up a lot of space. Essential resources are also expended through training, support, and maintenance. These types of additional factors are therefore also of interest. When considering improved seed sampling methods, it is not a trivial matter to balance multiple factors and outcomes against objectives.

For example, automated lysis is sought out or investigated many times in situations where a relatively large number of seeds are to be sampled within a limited time. However, automation not only introduces cost and complexity, but also real results. Therefore, there is reason to develop new non-automated seed sampling methods that meet as many of the above-listed goals for seed sampling as possible.

【Content of invention】

Therefore, one embodiment of the present invention is to provide a device, method or system that improves the problems and deficiencies of the prior art.

Further, embodiments of the present invention include devices, methods or systems that:

a. Improve the acceptable throughput of collecting samples from multiple individual seeds;

b. Facilitate sampling such that the sampled seeds have a relatively high germination rate;

c. Promote relatively consistent, accurately measured useful quantities of samples;

d. Prepare samples for valid biochemical, genetic, or dominant assays;

e. Facilitating more efficient selection during plant evolution experiments;

f. Avoid sample contamination and cross-contamination between samples;

g. Facilitate sampling so that it does not prejudice biochemical, genetic or dominant testing;

h. Improve the accuracy, consistency and reliability of sampling;

i. Flexible application to various seed types and various sampling tasks;

j. is valid and stable;

k. capable of reducing the amount of site space and the number of samples that need to be analyzed and shipped for use in plant evolution experiments;

l. Reduce the materials needed for plant farm management;

m. Overall reduction of human resource requirements;

n. is non-complex;

o. is economical;

p. is portable;

q. is non-cumbersome or non-difficult with respect to training and application;

r. Reduce plant tracking and labeling workload;

s. Improve the ergonomics of the sampling process.

One embodiment of the present invention is a method whereby, without significantly reducing the germination potential or viability of the sampled seeds, the seeds are removed from the seeds by shearing or similar action using one or more blades. and collect a useful amount of tissue volume to single-sample seeds from one generation in a plant evolution experiment. The tissue is then processed to obtain one or more biochemical, genetic, or dominant characteristics of the seed, after which a decision is made whether to use the seed in further plant evolution experiments or other plant-based research Decide. This allows a decision to be made as soon as the seeds are mature in a particular generation, as opposed to harvesting said seeds, planting them in test plots, and subsequently passing scientific analysis (e.g. genetic tests for the presence of traits, said Traits affecting seed components such as oils, proteins, starches, or other such procedures) test immature plants. This enables an earlier decision as to which offspring will be selected to continue in the experiment. Further, this reduces the amount of land required for test fields. It essentially reduces the amount of land required to screen thousands of plants in a single plantation by identifying 10%-20% of the seeds needed for evolution. As a result, field sampling is not required because the determination is not based on plant tissue. This seed sampling technique allows rapid and efficient determination of which seeds contain desired genetic traits or other characteristics.

Another embodiment of the present invention is a method, device and system for efficiently obtaining useful quantities of samples and locations from seeds while maintaining a high probability of germination of the seeds.

Another embodiment of the invention prevents or minimizes contamination in sample extraction and processing.

These and other embodiments of the invention will become apparent upon reference to the appended detailed description and claims.

【Description of drawings】

Reference will now be made to several of the accompanying drawings and examples which are in addition to this description and which are hereby incorporated by reference into this description.

Figure 1 illustrates a single seed clipping apparatus and method according to one embodiment of the present invention.

Figures 2A and B illustrate a single seed location and clipping tool, and sample indexing system, according to another embodiment of the present invention.

【Detailed ways】

A. Overview

In order to better understand the present invention, how various aspects of the present invention are implemented will now be described in detail. It should be understood that these examples are only a few forms the invention can take, and are not intended to limit the invention.

Frequent reference will be made to the drawings. Reference numerals and/or letters will be used to indicate certain parts and locations throughout the drawings. Unless otherwise indicated, the same reference numerals will be used to designate the same parts or positions.

The context of these particular examples will be with respect to corn kernels. It should be understood, however, that this example is only intended to illustrate one application of the invention. The invention can be used with other seeds and other objects. Size ranges may vary with object properties. As will be appreciated by one of ordinary skill in the art, embodiments of the present invention will be used with seeds of a size that is convenient for sampling. Some seeds are very fine and small, some resembling grains of dust or salt, while others are extra large and hard, such as the seeds from the Lodoucea maldivica palm, which weigh 20 to 24 pounds. One of ordinary skill in the art recognizes that the size and weight of the seeds intended for use with embodiments of the present invention must be convenient for sampling with the devices of the present embodiments. Such seeds include, but are not limited to, many agriculturally important seeds, such as seeds from corn, soybean, Brassica species, canola, cereals (such as wheat, oats, or other grains), and various plants and Seeds of ornamental plants. Similar applications will become apparent from this example class, and variations that would be obvious to one of ordinary skill in the art will be incorporated.

Reference will be made to samples taken from seeds. Sampling methods may refer to different terms such as sampling, cutting, clipping, slicing, cutting, clipping or removing a sample. The samples that have been taken can also be referred to using different terms, eg, seed sample, seed tissue sample, seed fragment, seed section, seed flake, seed clipping, and seed part.

B. General approach

In one embodiment, the present invention utilizes mechanical shearing of seeds to create seed tissue samples for laboratory testing. The seeds are manually grasped, positioned, and desired portions of the seeds are clipped and placed into test tubes or other containers for testing. The remainder of the seed is likewise collected and marked for possible future selection and planting purposes. Seed viability after sampling is a choice for the user and will be affected by where the sample is taken from the seed.

The present invention can be used to obtain seed samples which are then used for various analyses, such as DNA, RNA, protein, spectroscopic, and assays based on seed composition, where laboratory results determine which seed will be used for breeding or other testing sexual purpose.

The method addresses objectives (a)-(f) of the background art enumeration in at least the following manner.

(a) Viability

The shearing action allows for the separation of snips, chips, flakes or other individual pieces from the seed without crushing, tearing or otherwise destroying the viability of the sample. By using a reasonably sharp cutting edge, and taking advantage of the mechanical advantage of a shearing tool, an undamaged, often relatively small portion of the seed is removed, leaving the desired structure and tissue to germinate the seed.

(b) Sample size

The selection of clipping tools and methods can be commensurate with the size and type of seed, so that the required minimum sample size can be obtained for testing, but without removing too much (for example, an amount that may substantially reduce the risk of germination probability), likewise, Tool selection and manipulation relative to the seed tool allows flexible and adjustable sample sizes to be taken. Although this may involve manual handling of tools and/or seeds, it has been found to provide a practical, easily taught, acceptably accurate and rapid method of obtaining samples for several applications involving seed samples.

(c) Sample location

Similar to sample size, selecting an appropriately sized and configured clipping tool allows control over where on the seed the sample is taken. Again, while this can involve some manual effort (eg, grabbing, locating, and/or orienting the seed; and operating a hand-held clipping tool), it represents a practical approach to individual seed sampling for a certain seed sampling application.

(d) Throughput

Clipping samples from a single seed, one at a time, seems to defeat the throughput goal. However, as discussed above, production volume is not the only consideration in seed sampling for plant breeding. It has been found that even hand-operated clipping tools and manual processing of seeds can provide a level of throughput suitable for plant breeding applications; especially while taking into account listed objectives (a)-(f) and practical circumstances, including but not limited to cost, complexity , portability, training, support, maintenance, and more. Whereas automated solutions require large systems that take up considerable space and are very expensive, the sample system of the embodiments is simple, cheap, highly adaptable depending on the amount of seed to be sampled and the time available for such sampling. Additionally, staff with extreme training can be rapidly added for sporadic hours when large numbers of samples are required. This prevents the need for an additional large expensive system that would sit idle for long periods of time.

(e) Avoid pollution

As mentioned, clipping samples minimizes or eliminates the risk of contamination, including cross-contamination between samples. For example, samples tend to be monolithic. Therefore, there is a lower risk of residual flakes from previous samples. Again, clipping typically does not produce any dust, debris, or other very small particles that are difficult to discern or remove between samples. This compares to eg drilling, milling, grinding, rasp or drilling tools which tend to create such dust or small particles.

Although there is direct contact between the clipping tool and the seed, the risk of contamination between samples is low. Steps to further reduce this risk can be used. For example, the snipping tool can be cleaned between samples. However, such cleaning is not strictly required. Some of the seeds were fairly hard and when sheared, produced no splinters or dust and left no residue on the shearing blades. Additionally, many laboratory tests will be able to identify small amounts of contamination caused by earlier samples.

(g) Tracking samples

As previously indicated, mechanical shearing can involve manual steps (eg, the seed can be manually grasped, positioned, and the desired portion of the seed sheared with a manual actuation tool). However, processing of samples (and sampled seeds) may include steps that allow for higher throughput and the ability to efficiently and correctly track and correlate samples and/or sampled seeds.

For example, a clipped sample can be placed into a test tube or other container for storage and/or testing. The remainder of the seed can also be collected and tracked for possible future selection and planting purposes.

Various tracking methods known in plant breeding can be used for tracking. A few non-limiting examples include barcodes, RFID tags, printed or handwritten labels, and the like. Storage containers such as indexed seed trays or similar devices can be used for efficient organization and tracking.

Examples of how the specified objectives and other considerations can be factored into seed sampling by clipping are further seen from the specific exemplary embodiments set forth below.

C. Specific examples

1. Example 1 - Individually Clip Samples Using a Handheld and Operated Clipping Tool (Figure 1)

a) device

Figure 1 shows a shearing tool held and operated relative to a corn seed. The clipping tool is a fairly simple, relatively inexpensive, highly portable tool for individual seed samples.

In this embodiment, the clipping tools are conventional dog or cat scissors 330 . Such scissors are commercially available finished products from various sources. Opposing jaws, each with a sharpened blade, are used to snip a sample from the corn seed, eg, from the crown of the seed, in order to obtain a useful amount of germ, eg, for genetic testing.

An example of this pet nail clipping tool is the fairly ergonomic, easy to use and inexpensive item #743C (heat treated steel with safety stop bar serving as a selectable depth for seed size only) from Millers Forge, 1411 Capital Avenue, Plano, Texas 75974, USA. These nail clippers are suitable for various styles and sizes (such as large dogs, cats, etc.). The typical style indicated in Figure 1 has opposing jaws with concavely curved sharp edges that surround the seed and apply cutting pressure from different sides, including opposite sides of the seed. This tends to avoid applying pressure from only one side, or a dominant amount of pressure from one side. This prevents damage to the seed during cutting. For example, cutting a seed with a knife that holds the seed to a surface tends to exert pressure from one direction. This has the potential to damage, crack, crush or otherwise affect seed viability. Likewise, the inwardly facing concave blades give a de facto positioner for the seed relative to the blades, at least along one axis. It is however possible to use a single blade cutting tool.

A shearing tool 330 for corn seeds must be strong enough to handle the reaction force attempting to move the blade through corn seeds, which tend to be relatively hard seeds. Certain professional grade dog nail clippers should be sufficient. Many of these nail clippers utilize steel (even surgical tool steel) and have strong jaws, handles and pivots.

Several styles of animal nail clippers exist. Most nail clippers are configured to assist users who cannot confirm the basic accuracy of the cutting plane relative to the pet's nail, since it is important not to cut too close to the animal's flesh. The depth-stop feature on many pet nail clippers is designed to prevent injury to animals by limiting the depth of cut nails, which is particularly useful in making the clipper suitable for seed sampling. The depth stop allows the user to adjust the size of the sample taken from the seed and allows for consistent repeatability in taking seed samples from seeds of the same size. Most nail clippers are configured to avoid clipping, tearing or pain on parts of the animal. In a similar sense, seed sampling requires substantial precision in volume and location, as well as a cut that provides relatively easy cleaning. Thus, these types of shears also resist tearing, crushing, or turning of the seed when cutting, so as to promote a clean, precise, consistent cut.

Figure 1 shows a scissor style dog nail clipper. It works like a pair of scissors. There are two cutting blades like scissors, and when the handles are pushed together, the large pieces wrap around and cut through the nail. These scissor style nail clippers are not intended for larger thick nails but smaller thin nails for lighter jobs such as very small dogs or cats and bird claws. Scissors are a tool that takes advantage of mechanical advantages while requiring less force to cut. They are state-of-the-art dual-bar mechanisms with a pivot used as a fulcrum. Cutting is by applying a localized shear force at the cut site, which exceeds the shear strength of the material.

Plier-style dog nail clippers are similar to scissor-style, but tend to be stronger (eg, better suited for larger pets' nails). They tend to work in the same way as, say, pruning shears. When the handles are squeezed together, the two kerf blades encircle and subsequently cut the nail. Many groomers love these dog nail clippers because they make it easy to determine exactly where the blades are to cut the nails. The pliers style are basically heavy duty dog nail clippers. Some pliers-type cutters cut through the object being cut through indents and wedges, as opposed to the shearing action of a scissor-type tool. Pliers type cutters can be pointed out for larger and harder seeds.

As can be appreciated, characteristics of the seed being sampled can affect the style of scissors used. A pincer style or a more robust scissor style is indicated for corn seed. The size of the opening in the blade can vary. An appropriate size should be chosen for the type of seed to be sampled. Seeds that are much smaller than corn for example will require smaller scale sized shears; use the sizes possible on smaller animals such as cats, birds, rabbits, etc.

There are also guillotine-style dog nail clippers, which are also common and widely commercially available, including from sources such as the Millers Forge Company (see, eg, Model 744C Pet Nail Trimmer). A dog nail is inserted into the hole located in the crown of the adjuster. The handle is squeezed and the blade moves linearly through the nail. It can be heavy duty to allow insertion of thicker nails into the pilot holes. However, this shear will work acceptably for many seed types and sizes.

However, other types or classes of cutting tools could equally be used for this seed sampling. Alternative embodiments will be discussed later in this application document.

b) Operation

(1) Pretreatment

The only pretreatment of seed 3 was that it was dehusked and separated from its ear.

(2) Orientation

Workers will manually manipulate the seed 3 into the desired orientation.

(3) Sample cutting

The operator of the tool 330 will be trained to manually grasp the top cap end of the seed 3 in one hand and insert the coronal end of the seed 3 between the opposing jaws with the jaws facing inward or towards the open position ( As shown in Figure 1). In this embodiment of tool 330, the facing blades are curved in a concave shape. The open area between them ( FIG. 1 ) acts in fact as a guide or locator for the seed 3 between the jaws. The operator then carefully positions the seed 3 relative to the cutting plane of the jaws so that the desired useful amount of the coronal end of the seed 3 is aligned with the cutting plane. The concave shaped blade promotes a clean cut and good control when cutting relatively small object seeds. The nature of the blade also provides the operator with a relatively good view of the seed in order to improve cut placement accuracy and sample volume removed.

The operator manually initiates the cutting of the seed 3 by folding the handle of the tool in his opposite hand. The operator utilizes the mechanical advantage of the handle in a controlled manner to converge the jaws through the seed 3 to cut or dissect the sample.

(4) Sample collection

Samples can be collected on the plate by hand, or into bullet-type test tubes, and transferred to the appropriate wells of a sample plate (see Figure 2B for an example of one type of sample plate or index tray 59). Alternatively, it can be used for the operator to clip the sample from the seed 3 and allow it to drop right down by gravity into the well of the tray 59 in Figure 2B.

In the examples given, the average size of samples from corn seed crowns was between 0.5 and 20 mg. Ability to vary the number of useful samples according to seed type and application. If corn seed viability does not need to be maintained after sampling, a sample size of 30 or 40 mg, or more, would be acceptable and readily obtainable using embodiments of the present invention.

(5) Collection of cut seeds

The sampled seeds can also be manually or otherwise moved into an index tray 59 or similar storage. By placing a sample or sampled seed in the same indexed position in each tray 59, the sampled seed can be linked to its sample. Labels, barcodes or other identifiers for each tray can be associated with tracking and maintaining correlation between samples and seeds. As noted, the identifier can be machine-readable to allow efficient electronic or computer storage and retrieval of such information.

This embodiment balances the various issues and factors associated with seed sampling in the manner described above with respect to the general methods described above. Controlled sample clipping has been shown to not significantly affect the germination potential of the sampled seeds. Controlled clipping can provide useful amounts of samples. It enables control over the size of the seed clipping or sample removal and the location of the tissue removed from the seed. It can be controlled in order to reduce the risk of removing too much sample. Contamination risk can be minimized or eliminated by obtaining seed clips with a clean cut

(6) post-processing

Sample testing can take place directly on the tray 59 or otherwise according to methods known in the art. After the test results are obtained, the samples and/or seeds can be recovered and prepared for shipment and use based on the test results of the samples (eg, an indication of the presence of a desired property or characteristic).

Biochemical, genetic or phenotypic testing of seed tissue samples can be performed. The cut seed can then be used in relation to the sample of cut seed after determining whether it contains the desired biochemical, genetic, or dominant trait.

2. Example 2 - Samples were individually cut with a cigar-shaped multi-blade cutter (FIG. 2A)

a) Device (Figure 2A)

Figure 2A shows a single seed sample tool 300 that is hand or bench mounted. The funnel-shaped housing or body 302 holds the curved cutting blades 310 of a conventional commercially available cigar cutter 309 (eg, three tangentially moving blades with scissor-type handles), which will be referred to as the cutting plane. Some cigar cutters have more than three blades.

Such cigar cutters are commercially available. Examples are the XiMTX Multi-Tool available from Xikor of Kansas City, MO 64102 and the Model 8173B Scissor Cigar Cutter available from Cubanoz.biz of Miami Beach, FL 33119.

A hinged cover 306 attached around the body 302 has at its center a rubber or flexible gasket 308 sized to accommodate a single corn seed 3 when the seed 3 is pushed into the center of the gasket 308 . The geometry of the cover 306 and gasket 308 is such that a desired amount of seed 3 (here the crown) extends through the cutting plane of the blade 310 when the cover 306 is pivotally downwardly abutted against the body 302 . The cutting tool 309 is then operated to cut off parts (or samples) of the seed 3 .

b) Operation

(1) Pretreatment

The only pre-treatment of the seed 3 is that it is dehusked and separated from its ear so that it can be placed into the washer 308 manually.

(2) Separation

As can be appreciated, workers will typically track the origin of individual seeds 3 so that correspondence between the various seeds, their origins and samples can be maintained.

(3) Orientation

A worker will manually manipulate the seed 3 into the gasket 308 in the desired direction.

(4) Sample cutting

When the handle 312 is removed, the blade moves so as to provide an opening in which the seed 3 can be placed. When the lid 306 is closed, the seed 3 is placed and positioned in the opening. Figure 2A shows cutting tool 309 with handle 312 moved to a cutting position that closes the gap between blades 310 in the cutting plane. The seeds 3 in the openings in the cutting plane will be cut in order to separate parts or samples of the seeds 3 .

As in the embodiment of Fig. 1, this embodiment has a knife edge surrounding the seed. When converging, the blades initiate cutting action in unison, often along a single cutting plane from multiple directions, to even cut precisely and cleanly and prevent damage to the seed.

As mentioned above, it is preferred that the sample is isolated in a size sufficient for accurate biochemical, genetic or phenotype testing, and small enough to minimize the effect on the germination potential of the seed 3 .

(5) Sample collection

In the examples given, the average size of the samples lies between 0.5 and 20 mg. When cutting a sample, the square outlet tube 314 of the cutting tool 300 can be placed into (or on) an appropriate well in the sample plate to deposit the sample directly into the sample plate. The square end of the 314 fits snugly into a square well for precise delivery. By simply moving the tool 300 to the next sample plate well, the cut of the next seed 3 can be deposited into the next sample plate well, and so on.

(6) Collection of cut seeds

The remainder of the seed 3 will be held in the gasket 308 . The seeds 3 can be directed into individual wells located in the appropriate seed plate, at the same or related well positions corresponding to the corresponding samples in the sample plate.

This embodiment balances the various issues and factors associated with seed sampling in some ways similar to the previous embodiments and in some different ways. However those factors are balanced so as to achieve at least the following goals. Controlled sample cutting has shown that the control does not significantly affect the germination probability of the sampled seeds. Controlled cutting can provide a useful amount of sample. It can be used to control the size of the seed clipping or sample removal and to control the location of tissue removal from the seed. It can be controlled to reduce the risk of too much sample removal. One form of pre-sampling seed orientation is disclosed. It uses a receptacle to position the seed for controlled cutting with a blade. The arrangement also reduces the risk of contamination of the sample. Some configurations allow for quick and accurate placement of samples into different locations in the container to improve sampling throughput and sample collection and storage.

(7) post-processing

Biochemical, genetic or phenotype testing of samples can be performed as described above. Thus, the cut seed can then be used in relation to the sample of cut seed after determining whether it contains the desired biochemical, genetic or dominant trait.

This embodiment also addresses the specified objectives (a)-(f) as described in the general process example above.

D. Alternative and Alternative Implementations

As those of ordinary skill in the art will appreciate, the embodiments of the invention disclosed herein are illustrative only and not exhaustive of the forms in which they can be obtained. Variations obvious to those of ordinary skill in the art are intended to be within the scope of the invention and its embodiments. Some examples are set forth below.

1. Device

The dimensions, structures and materials of the components used in the exemplary embodiments can be changed as needed and desired.

Optionally, a number of manual or possibly heavy-duty clipping tools can be incorporated in place in a particular style of dog nail clipper 330 or cigar clipper 300 . Embodiments include, but are not limited to, off-the-shelf or modified versions of cutting equipment, such as ring scissors or angle scissors style snipping tools, tin snips, pipe cutters, pair of shears, pill snips, or wire cutters. The blade is usually sharp, but usually need not be dangerously sharp. Most tools of this type provide sufficient mechanical advantage and cutting action for sample removal.

Additionally, two or more moving blades of the embodiment in Figures 1 and 2A-B can be used. Also, a single-edged cutting blade with a stationary backplate or a stationary edge can also work.

As can be appreciated, many tools are available for cutting or shearing a sample from the seed. Ability to use a number of reasonably sharp tools of mechanical interest.

The raising system can sense with a sensor (eg, an optical sensor) when the seed is in position relative to the cutting blade, and then trigger an actuator that will automatically close the blade and cut the sample.

2. Method

The precise method steps can be varied as needed and desired.

3. Orientation

Some assisted orientation is possible before clipping the seeds. For example, some types of templates or receptors are capable of mechanically confining seeds in a desired orientation that is repeatable between successive seeds. It should also be noted that the orientation of at least some of the seeds can be done manually by how they are inserted therein (eg, receptacles, holes, cups, or other structures). In some cases, the geometry of this structure helps to orient the seeds. This is especially effective for seeds that have an asymmetric structure but compatible shape, maize is one such example.

4. Post-processing

Optionally, a post-sampling seed treatment or similar substance can be applied across the seed area where the sample was taken.

For example, a substance such as paraffin can be placed or coated on the cut area of the cut seed 60 . There are also commercially available grain protection layers (eg, the Log-Gevity ^™ product from ABR Products, Inc., Franklin, Wisconsin, USA) that can be used to prevent intruders from entering the cut seed 60 .

Another example would be to use a seed treatment such as Lockout ^™ (Becker Underwood Company) to protect nutrients from erosion from the seed.

Optionally, one or more substances can be applied to the sampled seed after sampling. Examples include, but are not limited to, pesticides, fertilizers or growth enhancers or antifungal agents.

A specific example is bentonite, a natural substance with antifungal properties. Such substances can be used to increase the probability of germination and reduce pathogenic attack on the cut seeds 60 . Commercially available chemical seed treatment fungicides include Captan ^™ , a broad spectrum fungicide from Drexel Chemical Company, Memphis, TN, USA; and Apron®, from Syngenta, Greensburg, NC, USA. ^TM (metalaxyl) and Maxim ^TM (fludioxonil) fungicides.

5. Analytical techniques

As will be appreciated by those of ordinary skill in the art, the method by which a sample is processed to derive biochemical, genetic or overt information can comprise virtually any method known in the art. Many methods are well documented and well known.

6. Application

a) Seed type

The exemplary embodiments and invention are not limited to corn seeds, but can be used with virtually any seed. Soybean and canola seeds are two other examples. Other seeds of interest include, but are not limited to, seeds from canola, sorghum, wheat, sunflower, Brassica species, rice, oats and other cereals and other agriculturally important crops.

b) purpose

Embodiments of the present invention find application in a wide variety of laboratory assays and procedures, and can be used in all aspects of plant research. Some but not all of the ways in which the present invention can be applied include DNA and RNA extraction and testing procedures, genotyping, selection for genetically improved seeds against non-genetically improved seeds, marker identification, testing for the presence of foreign species, Spectroscopy, food research, oil chemistry, and protein biochemistry. This embodiment is not intended to be limiting in any way, but is an example of the method by which embodiments of the present invention find use.

Claims (28)

1. the method for the seed of sampling, it comprises:

A. manually with the described placing of seed in predetermined orientation;

B. by manually impelling the part of one or more blade movements by described seed to remove measurable sample from described seed.

2. method according to claim 1 is wherein removed the remarkable minimizing that measurable sample can not cause described germination possibility from described seed.

3. method according to claim 1, wherein said seed is a corn seed.

4. method according to claim 3, the sample on the wherein said seed are to take from the bizet end of described seed.

5. method according to claim 1, manually be defined in described seed predetermined orientation the wherein said manually described placing of seed being comprised in predetermined directed step.

6. method according to claim 1, the motion of wherein said one or more blades comprise scissor action or pliers action.

7. device that is used for single kind of sub sampling, it comprises:

A. instrument, it comprises

I. the end of working, it comprises blade;

Ii. handle, it is operably connected to described work end to impel described blade motion;

B. container, it is used to hold by described instrument from the isolated sample of described seed.

8. device according to claim 7, wherein said device further comprise makes described seed with respect to described blade location and directed mechanism.

9. device according to claim 8 wherein saidly is used to make the described placing of seed and directed mechanism to comprise receptacle, and described receptacle has the geometric configuration that mechanically described seed is limited to described position and described predetermined orientation.

10. device according to claim 7, wherein said instrument comprise a kind of instrument that is selected from described group, are made up of following for described group:

A. the pet nail of scissors action is cut;

B. the pet nail of pliers effect is cut;

C. tinsmith snips;

D. pipe cutter;

E. pill is cut;

F. combination pliers; And

G. cigar is cut.

11. device according to claim 7, wherein said device further comprises the container that is used to collect each seed that was sampled.

12. the method from single kind of sub sampling seed tissue, it comprises:

A. utilize hand-held and cutting tool operation that the relative less part of the described seed remainder with described seed is separated, wherein said step of separating is fit to provide measurable relatively sample of seed.

13. method according to claim 12, wherein said division step is further adapted for the remainder that does not destroy described seed.

14. method according to claim 12, wherein said cutting tool comprise one in following:

A. pet nail is cut;

B. cigar is cut.

15. further comprising, method according to claim 12, wherein said method collect described sample to prevent sample contamination.

16. method according to claim 12, wherein said method further comprise in described sample and the seed one or two are enrolled directory system.

17. method according to claim 12, wherein said method further comprise described sample is used to comprise application biochemical, test gene or dominance.

18. method according to claim 17, test wherein said biochemistry, gene or dominance comprise one or more in following:

A.DNA extracts;

B.RNA extracts;

C. genotyping;

D. be that transgenosis is chosen seeds to not genetically modified Tl seed;

E. mark identification;

F. accidental the existence tests;

G. spectroscopy;

H. food processing;

I. food research;

J. oiling is learned; And

K. protein biochemistry.

19. method according to claim 18, wherein said method comprise that further the test that utilizes described biochemistry, gene or dominance makes in the plant evolution experiment decision that the remainder about described seed uses.

20. method according to claim 19, wherein said seed comprise corn seed, Canadian rape (Canola) seed or soya seeds.

21. method according to claim 12, wherein said method further are included as continuous a plurality of single seed repeating step.

22. the method for a plurality of single seeds of sampling, it comprises:

A. collect one group of single seed of known identities or origin;

B. in the following manner the single seed from described group is sampled continuously:

I. by manually starting cutting action the sample of consumption is arranged from the precalculated position clip on the described seed;

Ii. do not produce fragment, dust or particle;

C. follow the tracks of each sample that is positioned at the unique index position.

23. method according to claim 22, the wherein said method of sampling does not cause remarkable influence for the described germination possibility that is sampled seed.

24. comprising usually, method according to claim 22, wherein said clip one or more blades are pressed in the described seed along single cutting planes.

25. method according to claim 22, wherein said sample account for the cumulative volume of described seed or the less relatively number percent of general assembly (TW).

26. further being included in, method according to claim 22, wherein said method carry out biochemical, test gene or dominance on each sample.

27. method according to claim 26, wherein said method comprise further that collection and tracking and corresponding sample are mutually related and are sampled seed so that select or plantation.

28. a non-lethal method of sampling, the described method of sampling are used for from some single corn seeds each obtaining the sample of consumption, to be used for physics, chemistry or genetic test, the described method of sampling comprises:

A. each corn seed is carried out clip:

I. less relatively clip;

Ii. from bizet or near bizet and away from plumule;

Iii. use hand-held manual cutting tool;

Iv. do not produce remarkable dust, fragment or pollutant; And

B. follow the tracks of each sample.

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