U.S. patent application number 12/220083 was filed with the patent office on 2008-11-20 for identification of seeds or plants using phenotypic markers.
Invention is credited to Stefan A. Bledig, Vergel C. Concibido, Timothy W. Conner, Greg A. Penner.
Application Number | 20080289061 12/220083 |
Document ID | / |
Family ID | 26952502 |
Filed Date | 2008-11-20 |
United States Patent
Application |
20080289061 |
Kind Code |
A1 |
Penner; Greg A. ; et
al. |
November 20, 2008 |
Identification of seeds or plants using phenotypic markers
Abstract
Utilizing phenotypic markers in seeds or plants to allow
qualitative detection of a proprietary trait in the harvest, to
allow a quantitative calculation of the amount of the trait, and to
facilitate the calculation and collection of fees for the trait.
The phenotypic markers of the seeds can be the seed coat color, and
said seeds can be homozygous or heterozygous for the phenotypic
difference of seed coat color. Commercial cultivars of seeds with
the phenotypic difference of seed color may be grown to include
several different seed colors. Trait fees may be assessed on all
grain with the proprietary trait, whether the grain was produced
from purchased seed or from seed saved from a previous harvest.
Inventors: |
Penner; Greg A.; (Guelph,
CA) ; Bledig; Stefan A.; (Chesterfield, MO) ;
Conner; Timothy W.; (Chesterfield, MO) ; Concibido;
Vergel C.; (Maryland Heights, MO) |
Correspondence
Address: |
HOWREY LLP
C/O IP DOCKETING DEPARTMENT, 2941 FAIRVIEW PARK DRIVE SUITE 200
FALLS CHURCH
VA
22042
US
|
Family ID: |
26952502 |
Appl. No.: |
12/220083 |
Filed: |
July 21, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10071272 |
Feb 8, 2002 |
7402731 |
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12220083 |
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60267551 |
Feb 9, 2001 |
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60327801 |
Oct 9, 2001 |
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Current U.S.
Class: |
800/266 |
Current CPC
Class: |
C12N 15/8274 20130101;
A01H 5/10 20130101; A01H 1/02 20130101; C12N 15/8209 20130101; A01H
1/04 20130101 |
Class at
Publication: |
800/266 |
International
Class: |
A01H 1/04 20060101
A01H001/04 |
Claims
1-23. (canceled)
24. A method of generating seeds of a cultivar that are homozygous
and heterozygous for coat color, the method comprising: mixing
herbicide resistant, secondary colored seed coat seeds, with
non-herbicide resistant primary colored seed coat seeds to form a
first seed mixture; planting said first seed mixture in a field;
bulk harvesting the field to produce a second seed mixture;
planting the second seed mixture; spraying the field with the
herbicide for which herbicide resistance exists for the herbicide
resistant seeds; harvesting the remaining seed to produce a
herbicidally culled seed mixture; separating, in the herbicidally
culled seed mixture, seeds with primary colored seed coat from the
other seeds, through the use of a color sorter; and retaining the
separated primary color seeds, which are homozygous and
heterozygous for coat color.
25. The method of claim 24, wherein the cultivar is soybean, canola
or wheat.
26. The method of claim 24, wherein the cultivar is soybean.
27. The method of claim 26, wherein the primary seed coat color is
yellow.
28. The method of claim 24 wherein the secondary seed coat color
can be determined by measuring the total light reflectance with a
near-infrared spectrophotometer for wavelengths from 550 to 650
nanometers or by optical scanning technology.
29. The method of claim 26, wherein the secondary seed coat color
is homozygous black.
30. The method of claim 29, wherein the homozygous black seed
genotype is RRiiTT.
31. The method of claim 24, wherein the ratio of secondary coat
color seed to primary coat color seed in the first seed mixture is
about 10/90 to 90/10.
32. The method of claim 24, wherein the ratio of secondary coat
color seed to primary coat color seed in the first seed mixture is
about 50/50.
33. The method of claim 24 wherein the first seed mixture is
planted on a field at least one hectare in size.
34. The method of claim 24 wherein the second seed mixture is
planted at a higher seeding rate than the first seed mixture.
35. The method of claim 24 wherein the second seed mixture is
planted at double the seeding rate of the first seed mixture.
36. The method of claim 42, wherein 0.1% to about 10% by weight of
the separated primary coat color seeds are mixed with generic seeds
containing said trait of interest.
37. The method of claim 24, wherein the separated primary coat
color seeds are replanted once to produce an additional generation
of seeds that are homozygous and heterozygous for coat color.
38. The method of claim 24, wherein the separated primary coat
color seeds are replanted up to five times to produce additional
generations of seeds that are homozygous and heterozygous for coat
color.
39. The method of claim 36, wherein 0.1% to about 10% by weight of
the separated primary coat color seed is mixed with said generic
seeds to prevent the sorting out of seeds that are homozygous and
heterozygous for coat color.
40. The method of claim 29, wherein the genotype for the secondary
seed coat color comprises the ii genotype.
41. The method of claim 26, wherein the separated primary color
seeds comprise the ii or Ii genotype.
42. The method of claim 24, wherein said non-herbicide resistant
primary colored seed coat seeds comprise a trait of interest.
43. The method of claim 42, wherein the trait of interest is a
genetically modified trait.
44. The method of claim 24, wherein the secondary colored seed coat
seeds are herbicide resistant by virtue of comprising a genetically
modified trait.
45. The method of claim 44, wherein the genetically modified trait
is glyphosate resistance.
46. The method of claim 45, wherein the herbicide is glyphosate.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/267,551, filed Feb. 9, 2001, and U.S.
Provisional Application No. 60/327,801, filed Oct. 9, 2001.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention is generally directed to a method of
using phenotypic markers in commercial seed or plant cultivars
containing proprietary traits, without actually physically linking
the marker to the presence or absence of the proprietary trait, to
facilitate the identification of the harvested grain, and further
allowing a collection of fees for the proprietary traits based on
the presence of the marker in the harvested grain.
[0004] 2. Background
[0005] The introduction of genes into plants, either through
genetic transformation or through marker assisted breeding, results
in the development of cultivars with improved characteristics.
These improvements include such characteristics as enhanced
agronomic performance or value-added end-use properties.
[0006] It is well known in the art that a phenotypic difference
such as leaf color or seed coat color may distinguish a plant or
seed line from other similar lines. An alternate seed coat color
has been incorporated into the genes of sunflower seeds in U.S.
Pat. No. 4,627,192 (Fick). However, many plants that have been
improved through technological manipulation may not be visually
distinguishable from unimproved plants. The lack of easily
distinguishable characteristics makes it difficult to collect a fee
for the proprietary trait or traits, or to otherwise track
harvested grain containing the trait.
[0007] The current methods of generating seed cultivars with a
phenotypic leaf color or seed coat color are labor intensive and
impractical. The flowering habits of the plants constrain the
process. Hand pollination is expensive and time consuming. The use
of genetic male sterility systems requires complex methods of
female seed increase, thus placing significant constraints on plant
breeding and necessarily results in hybrids that segregate for male
fertility.
[0008] Thus, there exists a need and desire for a simple method of
detecting the presence of proprietary traits in plants, seeds, or
harvested grain to facilitate collection of fees for the
proprietary traits. A method of efficiently generating large
quantities of seed cultivars with a phenotypic leaf color or seed
coat color is also desired.
SUMMARY OF THE INVENTION
[0009] Commercial seed of cultivars containing proprietary traits
are produced in a normal manner. Seeds useful in the present
invention may contain one or more proprietary traits. Prior to the
sale of such proprietary seed to growers, generic or proprietary
seed containing a phenotypic marker, such as the black seed coat
color in soybeans, are mixed with the seed containing the
proprietary trait at a certain low percentage between about 0.1%
and 10% by weight and preferably about 0.5% to 4% by weight of the
total seed mixture. Upon the purchase of the seed mixture with
phenotypic markers, a grower would agree to pay a fee for the
proprietary trait in the grain, as indicated by the presence and/or
quantity of the phenotypic marker within the grain.
[0010] The generation of large amounts of hybrid (F1) seeds by the
present invention relies on the use of a combination of herbicide
resistance and seed coat color. One example of a herbicide
resistant soybean developed by Monsanto Company that is useful in
carrying out this invention is sold under the trademark Roundup
Ready.RTM., however any herbicide resistant soybean can be used. An
example of a seed coat color gene useful in the present invention
is the black seed coat color gene encoded by the i allele at the I
locus. This gene is expressed in a recessive manner in the seed
coat and is therefore expressed in a maternal manner in seeds.
[0011] The method of mixing generic seed with seed containing a
phenotypic marker improves the enforcement of contracts between the
trait proprietor and licensee and also improves the detection of
patent and or contract infringement. The present invention also
facilitates an end-point value capture system that could result in
the trait proprietor being more willing to allow newer technology
to be used by the licensee. The present invention enables end-point
value capture even if growers mix grain containing a proprietary
trait with grain not containing a proprietary trait. The present
invention also improves the trait proprietor's ability to collect
licensing fees from grower saved seed. Additionally, the present
invention allows the detection of the proprietary trait by enabling
the reappearance of the phenotypic marker in the subsequent
generation after attempts have been made to remove it from a seed
lot by physical sorting.
[0012] A plant seed mixture useful in the present invention may
contain primary colored seeds and secondary colored seeds from the
plant species of soybean, canola, or wheat. If soybeans are used,
the secondary seed coat colors may be black, brown, heterozygous
yellow, or determined by measuring the total light reflectance with
a spectrophotometer for wavelengths from 550 to 650 nanometers.
[0013] The secondary colored seeds may be generated by planting
homozygote black seed coat soybean plants in separate, alternate
rows and increasing the seed over several generations, or by
separating the black seed coat seeds after the first generation and
only propagating them. An additional method of generating secondary
colored seeds involves mixing herbicide resistant seeds with
non-herbicide resistant seeds; planting and growing the seeds; and
spraying the plants with a herbicide such that only a mix of
herbicidally culled seeds are left. The primary colored seeds are
then separated and retained from the mix.
[0014] A seed mixture containing primary and secondary colored seed
coat may be used in a method of identifying seed with a proprietary
trait by planting and growing the seed mixture; harvesting the
grain from the plants; and taking a sample of the grain to
determine the amount of phenotypical marker present. Licensing fees
may be calculated based on the amount of phenotypical marker
present in the grain. A grower may receive a voucher or rebate
based on the amount of marker present in the grain.
[0015] One skilled in the art may appreciate that this approach
facilitates or enables trait identification and fee collection from
grain produced from grain produced from proprietary seed, whether
the planted seed was purchased or saved from a prior harvest.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0016] The following terms and phrases are used herein and are
intended to have the following meaning: [0017] "end-point value
capture" is the assessment and collection of trait fees at the
point of grain delivery following harvest, generally a primary
elevator; [0018] "proprietary trait" is a trait for which a patent
position is held, and/or is managed as a trade secret by the
proprietor; [0019] "saved seed" refers to seed that is saved by a
grower for replanting in subsequent years; [0020] "licensing fees"
are equivalent to royalties paid by the user for the right to use
the proprietary trait; [0021] "licensee" is the purchaser of a
proprietary trait who has signed a contract with the owners of the
proprietary trait which governs terms and conditions for the use of
the trait, and the form of renumeration for the right to use the
trait; [0022] "trait proprietor" is the owner and/or seller of the
proprietary trait; [0023] "brown bag seed" is seed that has been
harvested by one grower and sold to another grower for the purpose
of planting and harvesting a crop; [0024] "elevator" is the primary
commercial delivery point for harvested grain; [0025] "seed coat"
is the remnants of the outer integuments of a plant flower, and as
such is genetically identical to the plant on which the seed is
borne; [0026] "outcrossing" is the fertilization of one plant by
another plant possessing a different genetic makeup for the desired
phenotypic marker; [0027] "outcrossed seed" is seed produced by a
plant which as been fertilized by another plant possessing a
different genetic makeup for the desired phenotypic marker; [0028]
"selfed seed" is seed produced by a plant which has been fertilized
by another plant possessing the same genetic makeup for the desired
phenotypic marker; [0029] "grain" is harvested seed sold for
commercial purposes; [0030] "grower" is the person responsible for
planting, maintaining and harvesting a crop; [0031] "visibly
detectable trait" is a trait that can be seen to be different
without a need for secondary analysis involving crushing of tissue,
or the extraction of any compound; [0032] "sorted seed" is the act
of physically differentiating seed based on observation of a
visibly detectable trait; [0033] "variety" is a grouping of plants
that are homogeneous and stable, and clearly distinguishable by at
least one phenotypic characteristic from all other groupings of
plants; [0034] "herbicidally culled seed" is seed which is selected
as a result of herbicide application; [0035] "F1 seed" is the first
generation of seeds produced; [0036] "F2 seed" is the second
generation of seeds produced by replanting all or part of the F1
seed either with or without incorporating other seed cultivars.
[0037] The present invention is directed to a method of using
phenotypic markers in proprietary seed or plant cultivars to
facilitate (1) detection of harvested grain containing the
proprietary trait and (2) determination of licensing fees.
[0038] In one embodiment, the method includes the use of phenotypic
markers in soybeans. Soybean seed coat genetics have been taught by
Nagai, Woodworth, and Williams in several publications over the
years, the contents of which are incorporated by reference. In
soybeans, the gene "R" encodes black seed coat color. "R" is
completely dominant to "r" which is the gene for brown seed coat
color. A second locus initially designated "C-c" but subsequently
designated "T-t", alters black to "imperfect black" and brown to
"buff". Woodworth demonstrated that this gene had a pleiotropic
effect. Tawny pubescent varieties "T" have black or brown seed
coats. Gray pubescent varieties "tt" have imperfect black or buff
seed coats. [0039] RT--Black seed coat [0040] rT--Brown seed coat
[0041] Rt--Imperfect black seed coat [0042] rt--buff seed coat In
addition, Williams found that there is a flower color gene, "W1-w1"
that can affect this as well. [0043] RtW1--imperfect black [0044]
Rtw1--buff The following genes are also know to affect soybean seed
coat color: [0045] r.sup.m--allelic to R(R>r.sup.m>r) [0046]
r.sup.mT--black and brown stripes in concentric rings around the
hilum [0047] r.sup.mt--hard to distinguish, but exhibits faint
rings of black and brown stripes in concentric rings around the
hilum. and [0048] O-o--a separate locus wherein the genotype [0049]
orT--produces a dark reddish brown seed coat Additionally, there is
the "i" locus which has at least four known alleles. [0050]
i--results in the seed coat colors described above [0051]
i.sup.k--results in a saddle shape pattern of colors localized
around the hilum [0052] i.sup.i--dark hilum--other colors
restricted to the hilum [0053] I--light hilum--color is restricted
to the hilum and intensity reduced Another locus k, now designated
as k2, was found in the variety Kurakake, [0054] ii k--saddle
pattern [0055] (i.sup.i, ik, I)--self black One skilled in the art
will appreciate that this locus appears to be some sort of
suppressor of more active forms of "i".
[0056] Seed color may be measured using a Technicon near-infrared
reflectance (NIR) spectrophotometer calibrated to determine total
light reflectance (optical density) from 550 to 650 nanometers.
This wavelength setting allows separation of yellow from brown from
black seeds. Alternatively, optical scanning technology can be used
to distinguish seeds on the basis of color. Both NIR and optical
scanning can be set up for high-throughput analysis.
[0057] Most commercial soybean cultivars exhibit a yellow seed coat
color. Soybean seeds with a black coat color occur in approximately
one in ten thousand seeds in nature. The black seed coat phenotype
is encoded by the presence of homozygous "ii". "R" and "T" are
present in commercial germplasm and "i" works by allowing the
expression of R across the entire seed coat. In commercial
varieties, "I" suppresses all color expression which results in a
light colored hilum and prevents color expression outside of the
hilum region. In one embodiment of the present invention, the black
seed coat soybean cultivars of the present invention are believed
to have the genotype, RRiiTT.
[0058] The seed coat is maternal tissue in that it is derived from
somatic tissue from the plant in which the seed is set. One of
skill in the art can recognize that the seed coat color reflects
the genotype of the mother plant, not the genotype of the seed
itself. Thus, it is possible to generate a seed with a completely
yellow seed coat which is homozygous for the black seed coat gene
"ii" and which will give rise to black seed coat seeds only as a
result of self-pollination or pollination by another homozygous ii
plant.
[0059] In another descriptive embodiment of the present invention,
the plant species is canola. Commercial canola grain generally have
black seed coats. Yellow seed coat types are known, and have been
related to increased oil and protein contents in Brassica napus.
Van Deynze and Pauls teach the genetics of canola seed coat colors,
the contents of which are incorporated by reference. The
inheritance of the yellow seed coat trait in canola appears to be
influenced by three recessive nuclear genes (a, b and c).
[0060] The presence of the dominant allele at the A locus is
sufficient for black seed coat color. The presence of the dominant
allele at either the B or C locus is sufficient for brown seed coat
color.
The following combinations give rise to black seed coats:
A-B-C-
A-bbC-
A-B-cc
A-bbcc
[0061] The following combinations give rise to brown seed coats:
aaB-cc aaB-C- aabbC- The gene combination needed for yellow canola
seeds is aabbcc.
[0062] To generate commercial seed of cultivars containing black
seed coat soybeans, also referred to herein as "bsc", bsc soybean
seeds could be mixed into commercial seed in at least the following
ways. In one embodiment of the present invention, a mix could be
generated in the field. Bsc soybean plants which are homozygous for
the `i` allele at the I locus would be planted in separate rows in
the same field as cultivars containing proprietary traits that are
being increased for commercial seed sale. A treatment would be
applied to the yellow seed coat rows that promotes outcrossing. In
a preferred embodiment, this treatment includes the application of
a higher concentration of the herbicide than normally applied. This
may cause a decline in male fertility which could result in higher
outcrossing rates. One skilled in the art can appreciate that the
number of generations necessarily depends on the outcrossing rate
and the required percentage of bsc in the seed mix.
[0063] A second illustrative embodiment of the mixing method of the
present invention includes a mechanical mix. A bsc soybean plant
would be crossed with a yellow seed coat plant. The bsc progeny
after several generations of self-pollination would be retained. A
known amount of this seed would be added to the yellow seed coat
variety. The number of generations required for self-pollination
would depend on the total amount of trait-containing seed that
would be commercialized and the required percentage of bsc in the
seed mix.
[0064] A preferred illustrative embodiment of the mixing method of
the present invention is a combination of the field mix and the
mechanical mix techniques. Bsc seed would be mixed in commercial
seed as described for the mechanical mix above, and bsc seed would
be grown together with the commercial cultivar in the last season
of seed increase prior to commercial sale.
[0065] The field mix results in the generation and maintenance of a
higher level of individual plants that are heterozygous for the bsc
gene. This is desirable because it would help to preserve the
presence of the marker phenotype despite selection against it. The
mechanical mix approach will have a lower cost of goods of
production. One skilled in the art will appreciate that the
combined approach optimizes the advantages of both systems.
[0066] In an additional embodiment of the present invention, a
certain proportion of the bsc seed is actually yellow when sold to
growers. In the first year's harvest, it will yield black seed and
will thus deter the practice of on-farm seed sorting between seed
purchase and planting. An alternate approach to this embodiment is
to incorporate a second dominant seed coat color gene in the black
seed coat line. In a preferred embodiment, this seed coat color
should only be expressed in the absence of the black phenotype
which is the homozygous black gene state. A brown seed coat color
could be used as the second seed coat color. F2 seeds derived from
F1 hybrid could be rapidly and cost-effectively color sorted using
a near-infrared spectrophotometer based on the presence of the
brown seed coat color. Seed would be increased from these hybrids
for a further generation (F3 generation). This could be done
without the need for additional sorting or with ongoing sorting out
of the homozygous yellow lines. The F3 generation would be used as
the bsc source which ensures a reliable level of bsc seed and a
sufficient proportion of heterozygotes to deter on-farm seed
sorting. In an additional embodiment of the present invention the
same approach as described above regarding the use of a second seed
coat color gene may be applied wherein selection of F2 seed derived
from heterozgyous F1 individuals would not be enabled by the
presence of a second seed coat color gene, but by the detection of
the color expressed in heterozygous individuals carrying the Ii
genotype in the maternal plant with the use of an NIR machine
described elsewhere.
[0067] In an additional embodiment of the present invention the
same approach as described above regarding the detection of
heterozygous individuals for use in a mechanical mix could be
enabled through the incorporation of a gene that results in an
altered plant phenotype, such as an altered leaf shape, enabling
the selection of heterozygous F1 plants in the subsequent
generation. This approach would involve incorporating the
selectable dominant phenotype in the black seed coat donor
line.
[0068] In an additional embodiment of the present invention the
same approach as described above regarding the detection of
heterozygous individuals for use in a mechanical mix could be
enabled by mixing a herbicide resistant black seeded line with a
yellow seeded line that is not herbicide resistant. The F1 plants
could be selected in the next generation by employing two steps.
First, the yellow seeds would be sorted from the black seeds using
a color sorter (this would include homozygous yellow, and
heterozygous yellow). Second, the yellow seeded plants would be
sprayed with a herbicide the following generation. Only the F1
heterozygous plants would survive. Seed from these plants would be
harvested and increased for a further two generations. In addition,
it is possible that outcrossing could be increased by applying a
low level of herbicide while the yellow seeded, non-herbicide
resistant plants are flowering.
[0069] In another embodiment, herbicide resistant and black seed
coat seeds would be mixed with non-herbicide resistant yellow seed
coat seeds randomly and planted on a large scale in the field. A
preferred range of the ratio of herbicide resistant seeds to black
seeds is 10/90 to 90/10, but any mix level can be used. Additional
preferred ratios are 30/70, 50/50, or 70/30. A preferred embodiment
of this invention regards large scale seed production, meaning at
least a hectare of seed, but the area of field used may be any
size, depending on the amount of F1 seed production desired. A high
planting rate would be beneficial, as it would result in a more
efficient use of space, and a higher level of outcrossing among
plants. Soybean plants outcross with each other within a row at a
rate of approximately 1%. If the outcrossing levels are lower, then
increasing the size of the first crossing block will
compensate.
[0070] The field would be bulk harvested, and replanted. A higher
seeding rate than the initial rate may be used, with a preferred
rate being double the initial seeding rate. The 1% outcrossed
plants, derived from pollen from the yellow conventional soybean
plants fertilizing the black seeded herbicide resistant plants, and
the pollen from the black seeded herbicide resistant plants
fertilizing yellow conventional plants would give rise to F1 seeds
that are black and yellow respectively. These would be replanted
along with all the selfed seed. This field would be sprayed with
the herbicide for which herbicide resistance existed in the
parental material. This spray would eliminate all the selfed yellow
conventional seed. The remaining seed would be harvested.
[0071] Because the seed coat color is recessive and maternally
inherited, the F2 seed derived from the F1 plants, would all be
yellow in color and the F1 plants that produced this F2 seed would
all be herbicide resistant. The F2 seed would be separated from the
selfed black, herbicide resistant seed through the use of a color
sorter, such as a Technicon near-infrared reflectance (NIR)
spectrophotometer calibrated to determine total light reflectance
(optical density) from 550 to 650 nanometers. The yellow seed would
be retained.
[0072] The yellow F2 seed could be increased a further generation,
or up to five generations, in the presence of further herbicide
resistance selection in order to increase the amount of seed
produced, and then could be mixed with seeds containing proprietary
traits as a segregating phenotypic marker. Yellow F2 seed could
also be directly used as a seed mixture, without further increases.
This strategy would be equally effective if performed with a mix of
non-herbicide resistant black seed lines with herbicide resistant
yellow seeded lines.
[0073] Licensing fees may be calculated and collected from
different entities who use or collect the seed. In one embodiment
of the fee calculation procedure of the present invention, the
growers would agree to pay part or all of the licensing fee owed
for use of the proprietary technology upon delivery of grain
containing the proprietary trait. Growers would agree to deliver
grain with the proprietary trait only to designated elevators that
have agreed to collect a fee for the proprietary trait. Growers
would agree not to sort grain in a manner that would prevent or
hinder detection of the marker phenotype or phenotypes. The license
terms could apply to all grain produced from certified seed and
grain from all subsequent generations. The grower may be issued a
voucher upon payment of the fee which can be redeemed at the
proprietor of the trait to receive discounts or other incentives on
subsequent seed or chemical purchases.
[0074] In another embodiment of the method of fee collection of the
present invention, the elevators would agree to collect a fee for
grain containing the proprietary trait and to remit a portion of
this fee to the proprietor of the trait. Elevators would also agree
to allow access to their facilities by representatives from the
proprietor of the trait to oversee the collection of trait fees
and/or to audit grain inventories for the presence of grain with
the proprietary trait. In an additional embodiment, the elevators
could have all of the client account information stored on
computers, and customized software would facilitate record keeping
and fee assessment. This information could then be forwarded to the
proprietor's computers via a network connection or the internet for
record keeping, billing of either the grower or the elevator, and
accounting purposes.
[0075] Seed sampling and testing protocols should be well known to
one of skill in the art as indicated in the commonly used reference
International Rules for Seed Testing 1999, available from the
International Seed Testing Organization, all of the contents of
which are hereby incorporated herein by reference. In an embodiment
of the present invention, visual seed sampling may be used as a
qualitative measurement standard. The grower would have a 0.25%
threshold as a bottom level for detection. If the grower exceeds
this threshold for a particular grain delivery to the elevator,
then he or she pays licensing fees based on the amount of grain
delivered. If the threshold is exceeded, but the grower protests
the positive result for bsc, then a more specific test will be
performed to determine if the grain contains the proprietary
trait.
[0076] In an additional embodiment, the threshold values may be
applied in a quantitative determination of the amount of bsc
present. Threshold ranges such as 0.25% to 0.5% vs. 0.5% to 0.75%
may be used to determine the proprietary trait fee based on the
amount of bsc delivered to the elevator.
[0077] In view of the above, one of skill in the art should
appreciate the usefulness of the above described method. Further,
one of skill in the art should recognize that the method of the
present invention may be applied to crops other than soybeans such
as canola. A mixture of yellow canola seeds with a black seeded
canola line carrying a proprietary trait would enable trait fee
collection and tracking of proprietary traits at the point of
delivery in a manner identical to what is proposed for soybean. The
blue aleurone of wheat and/or the purple seed coat color may also
be used a similar manner. Utilizing the methods of the present
invention, phenotypic markers may also be used with proprietary
corn and cotton. The use of phenotypic markers should be within the
skill of one in the plant genetic arts and the usefulness of the
present invention should be apparent to such a person.
[0078] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
GENERAL INFORMATION RELEVANT TO THE EXAMPLES
[0079] The following term is used in describing the following
examples:
[0080] "Roundup Ultra.RTM." is the trade name for a common
glyphosate herbicide
[0081] "OC" is outcrossing
Example 1
[0082] The following example outlines a protocol for determining
whether it is possible to enhance outcrossing in soybeans.
Roundup.RTM. is used in treatments 2-7 at the amounts
specified.
TABLE-US-00001 TABLE 1 Quantity of Roundup .RTM. Spray Schedule
Treatment 1 unsprayed check Roundup .RTM. may be used for weed
control Treatment 2 80 oz/acre 1-2 weeks before flowering Treatment
3 64 oz/acre Twice, once 3-4 weeks before flowering and once 1-2
weeks before flowering Treatment 4 96 oz/acre 1-2 weeks before
flowering Treatment 5 80 oz/acre Twice, once 3-4 weeks before
flowering and once 1-2 weeks before flowering. Treatment 6 80
oz/acre 1-2 weeks before flowering Treatment 7 80 oz/acre Twice,
once 3-4 weeks before flowering and once 1-2 weeks before
flowering. Treatment 8 unsprayed check Roundup .RTM. may be used
for weed control Treatments 1 to 5 contain black and yellow seeded
plants in separate rows Treatments 6 to 8 contain black and yellow
seeded plants in the same rows
Upon review of the above, one skilled in the art should recognize
that the above method of applying high doses of herbicide should
decrease male fertility and thus increase outcrossing.
Example 2
[0083] The following example provides evidence for the reduction of
male fertility in cotton plants resistant to Roundup.RTM.. Roundup
Ultra.RTM. was sprayed over the top of the plants four times at the
following intervals: 31, 45, 58, and 73 days post sowing. There
were three different plots of plants and each plot received a
different rate of Roundup Ultra.RTM. as follows: 0, 16, and 24
ounces/acre. Evaluation for male sterility was made 2 to 3 times
per week for 7 weeks. Ten blooms per plot were hand-pollinated on
each of 10 days. A fertility score of 1 to 5 was given to each
plant where a score of 3-5 is considered fertile and less than 3 is
considered sterile. Table 2 shows the results of this study.
TABLE-US-00002 TABLE 2 Fertility Fertility Fertility Score of Score
of Score of Days Post- 0 oz/acre 16 oz/acre 24 oz/acre Sowing
Roundup .RTM. Roundup .RTM. Roundup .RTM. 66 4.7 1.5 1.5 69 4.7 1.8
1.9 71 4.7 1.6 1.7 73 4.7 1.9 2.3 75 4.5 2.8 1.6 80 4.3 2.2 2.0 82
4.6 1.4 1.3 85 4.3 2.8 1.7 89 4.6 3.6 2.2 93 4.6 3.0 2.4 95 4.5 1.9
1.9 98 4.4 2.1 1.6 102 4.5 2.2 1.6 106 4.6 3.4 2.8 110 4.5 3.7
3.6
[0084] Upon review of the above, one skilled in the art should
appreciate how male fertility is reduced in a dicotelydenous plant
with high doses of herbicide and thus leading to higher outcrossing
rates.
Example 3
[0085] The following Table 3 shows the results of a model for a
field mix of seed showing the percent of bsc soybean seeds in
various generations of both the proprietor's commercial seed
harvest and the growers harvest using a particular generation of
the commercial seed. One row of bsc seed is planted between two
rows of yellow seeds which means that the ratio of yellow seed to
bsc seed is approximately 2:1 and an outcrossing rate of 5% is
used. Only the yellow rows of seeds are harvested to generate the
commercial cultivars.
TABLE-US-00003 TABLE 3 Column/Row (See Table 4) B C D E F
Commercial Seed harvested 6 Generation # II Ii ii ii (black) ii
(yellow) 7 1 95.00% 5.00% 8 2 91.44% 7.25% 1.31% 1.31% 9 3 88.59%
8.20% 3.22% 1.31% 1.90% 10 4 86.10% 8.53% 5.37% 3.22% 2.15% 11 5
83.82% 8.57% 7.61% 5.37% 2.24% 12 6 81.67% 8.48% 9.86% 7.61% 2.25%
Column/Row (See Table 5) H I J K L % BSC in grower's field Year 1
Year 2 Year 3 Year 4 Year 5 Generation # 7 1 0.00% 1.25% 1.88%
2.19% 2.34% 8 2 1.31% 3.13% 3.75% 4.06% 4.22% 9 3 3.22% 5.26% 5.89%
6.20% 6.36% 10 4 5.37% 7.50% 8.12% 8.44% 8.59% 11 5 7.61% 9.75%
10.37% 10.69% 10.84% 12 6 9.86% 11.97% 12.60% 12.91% 13.07%
The formulas for the above model are as follows in Tables 4 and 5
under corresponding rows and columns:
TABLE-US-00004 TABLE 4 A B C D E 1 BSC % in 0.33 field 2
Outcrossing 0.05 rate 3 4 5 Seed harvested 6 Generation # II Ii ii
ii (black) 7 1 =1 - (1*B2) =1*B2 8 2 =+B7 - (B7*B$2) + =+(C7*0.5) -
=0.25*(C7 - 0.25*(C7 - (B$2*C7)) (B$2*C7*0.5) + B$2*B7 + C7*B$2) +
(0.5*B$2*C7) 0.5*B$2*C7 9 3 =+B8 - (B8*B$2) + =+(C8*0.5) -
=0.25*(C8 - =+D8 0.25*(C8 - (B$2*C8)) (B$2*C8*0.5) + B$2*B8 +
C8*B$2) + (0.5*B$2*C8) + D8 0.5*B$2*C8 10 4 =+B9 - (B9*B$2) +
=+(C9*0.5) - =0.25*(C9 - =+D9 0.25*(C9 - (B$2*C9)) (B$2*C9*0.5) +
B$2*B9 + C9*B$2) + (0.5*B$2*C9) + D9 0.5*B$2*C9 11 5 =+B10 -
(B10*B$2) + =+(C10*0.5) - =0.25*(C10 - =+D10 0.25*(C10 - (B$2*C10))
(B$2*C10*0.5) + B$2*B10 + C10*B$2) + (0.5*B$2*C10) + D10
0.5*B$2*C10 12 6 =+B11 - (B11*B$2) + =+(C11*0.5) - =0.25*(C11 -
=+D11 0.25*(C11 - (B$2*C11)) (B$2*C11*0.5) + B$2*B11 + C11*B$2) +
(0.5*B$2*C11) + D11 0.5*B$2*C11
TABLE-US-00005 TABLE 5 F G H I J K L 1 2 3 4 % B in grower's field
5 Year 1 Year 2 Year 3 Year 4 Year 5 6 ii (yellow) 7 =SUM(B7:D7)
=+D7 =+H7 + =+I7 + =+J7 + =+K7 + 0.25*C7 0.25*0.5*C$7 0.25*0.25*C$7
0.25*0.125*C$7 8 =+D8 - D7 =SUM(B8:D8) =+D8 =+H8 + =+I8 + =+J8 +
=+K8 + 0.25*C8 0.25*0.5*C$7 0.25*0.25*C$7 0.25*0.125*C$7 9 =+D9 -
D8 =SUM(B9:D9) =+D9 =+H9 + =+I9 + =+J9 + =+K9 + 0.25*C9
0.25*0.5*C$7 0.25*0.25*C$7 0.25*0.125*C$7 10 =+D10 - D9
=SUM(B10:D10) =+D10 =+H10 + =+I10 + =+J10 + =+K10 + 0.25*C10
0.25*0.5*C$7 0.25*0.25*C$7 0.25*0.125*C$7 11 =+D11 - D10
=SUM(B11:D11) =+D11 =+H11 + =+I11 + =+J11 + =+K11 + 0.25*C11
0.25*0.5*C$7 0.25*0.25*C$7 0.25*0.125*C$7 12 =+D12 - D11
=SUM(B12:D12) =+D12 =+H12 + =+I12 + =+J12 + =+K12 + 0.25*C12
0.25*0.5*C$7 0.25*0.25*C$7 0.25*0.125*C$7
[0086] In view of the above, one of skill in the art should
recognize that the amount of black seed in the grower's field can
be approximated through several generations. One can also note how
many generations are necessary to generate a commercial seed
cultivar that actually contains black seeds generated through
outcrossing.
Example 4
[0087] The following Tables 6 and 7 show the results of a model for
a mechanical mix of seed showing the percent of bsc soybean seeds
in various generations of the proprietor's commercial seed harvest.
This example of the model is based on producing enough seed such
that a grower will harvest 3% bsc. The model predicts the amount of
black seeds needed to mix with a particular generation of the
commercial cultivar to obtain a mixture of seeds which will give
the desired grower harvest of bsc. The model also shows how the
number of black seed units produced varies depending on whether
black seeds are sorted out of the mix.
Tables 8 and 9 show the model for the generation of Tables 6 and
7.
TABLE-US-00006 TABLE 6 A B C D E F G H 2 BSC % 3% 3 4 Seed total
black yellow harvested 5 Generation # II Ii ii ii ii No sorting 6
F1 100% 7 F2 25% 50% 25% 0% 25% Mix rate 8 F3 37.50% 25% 37.50% 25%
12.50% 6.86% 9 F4 43.75% 12.50% 43.75% 38% 6.25% 6.40% 10 F5 46.88%
6.25% 46.88% 44% 3.13% 6.19% 11 12 13 Seed increase 14 # of seeds
Weight(kg) 16 g/100 seeds 0.00016 15 F1 20 0.0032 16 F2 1200 0.192
17 F3 72000 11.52 18 F4 4320000 691.2 19 F5 259200000 41472 20 21
22 23 Mechanical mix with a field mix 24 OC rate 5% total black
yellow 25 Generation # II Ii ii ii ii No sorting 26 F1 100% 27 F2
23.75% 50.00% 26.25% 0% 26% 28 F3 34.44% 26.19% 39.38% 26% 13.13%
6.53% 29 F4 38.94% 14.82% 46.25% 39% 6.87% 6.01% 30 F5 40.51% 9.35%
50.14% 46% 3.89% 5.72% 31 32 33 Seed increase 34 # of seeds Weight
(kg) 16 g/100 seeds 0.00016 35 F1 20 0.0032 36 F2 1200 0.192 37 F3
72000 11.52 38 F4 4320000 691.2 39 F5 259200000 41472
TABLE-US-00007 TABLE 7 I J K L M N O 2 3 4 5 Units produced Remove
black Units produced Select hetero's Units produced Select hetero's
Units produced 6 with mix with mix and blacks with mix with mix 7
Mix rate Mix rate Mix rate 8 6.72 12.00% 3.84 4.29% 10.75 48.00%
0.96 9 432 20.00% 138.24 3.60% 768.00 96.00% 28.8 10 26784 36.00%
4608 3.29% 50416.94 192.00% 864 11 12 13 14 15 16 17 18 19 20 21 22
23 24 25 Units produced Remove black Units produced Select hetero's
Units produced Select hetero's Units produced 26 with mix with mix
and blacks with mix with mix 27 28 7.05 11.25% 4.10 4.28% 10.76
48.09% 0.96 29 460.37 17.19% 160.80 3.67% 753.90 86.92% 31.81 30
29017.66 25.89% 6406.78 3.40% 48775.01 140.63% 1179.57
TABLE-US-00008 TABLE 8 A B C D E F 4 Seed harvested total black
yellow 5 Generation # II Ii ii ii ii 6 F1 1 7 F2 0.25 0.5 0.25 =+D7
- F7 =+D7 8 F3 0.375 0.25 0.375 =+D8 - F8 =+D8 - D7 9 F4 =+B8 +
0.25*C8 =+C8*0.5 =+D8 + 0.25*C8 =+D9 - F9 =+D9 - D8 10 F5 =+B9 +
0.25*C9 =+C9*0.5 =+D9 + 0.25*C9 =+D10 - F10 =+D10 - D9 11 12 13
Seed increase 14 # of seeds Weight (kg) 16 g/100 seeds 15 F1 20
=+B15*G14 16 F2 =+B15*60 =+B16*G14 17 F3 =+B16*60 =+B17*G14 18 F4
=+B17*60 =+B18*G14 19 F5 =+B18*60 =+B19*G14 20 21 22 23 Mechanical
mix with a field mix 24 OC rate 0.05 total black yellow 25
Generation # II Ii ii ii ii 26 F1 1 27 F2 =0.25*(C26 - =0.5*(C26 -
=0.25*(C26 - =+D27 - F27 =+D27 C26*B$24) + B26 - C26*B$24) +
B$24*B26 + C26*B$24) + 0.5* B$24*B26 0.5*C26*B$24 B$24*C26 + D26 28
F3 =0.25*(C27 - =0.5*(C27 - =0.25*(C27 - =+D28 - F28 =+D28 -
C27*B$24) + B27 - C27*B$24) + B$24*B27 + C27*B$24) + 0.5* D27
B$24*B27 0.5*C27*B$24 B$24*C27 + D27 29 F4 =0.25*(C28 - =0.5*(C28 -
=0.25*(C28 - =+D29 - F29 =+D29 - C28*B$24) + B28 - C28*B$24) +
B$24*B28 + C28*B$24) + 0.5* D28 B$24*B28 0.5*C28*B$24 B$24*C28 +
D28 30 F5 =0.25*(C29 - =0.5*(C29 - =0.25*(C29 - =+D30 - F30 =+D30 -
C29*B$24) + B29 - C29*B$24) + B$24*B29 + C29*B$24) + 0.5* D29
B$24*B29 0.5*C29*B$24 B$24*C29 + D29 31 32 33 Seed increase 34 # of
seeds Weight (kg) 16 g/100 seeds 35 F1 20 0.0032 36 F2 1200 0.192
37 F3 72000 11.52 38 F4 4320000 691.2 39 F5 259200000 41472
TABLE-US-00009 TABLE 9 G H I J K L M N O 1 2 BSC % 0.03 3 4 5 No
sorting Units Remove black Units Select hetero's Units Select
hetero's Units produced produced produced produced 6 with mix with
mix and blacks with mix with mix 7 Mix rate Mix rate Mix rate Mix
rate 8 =+H$2/ =+C17/ =+H$2/ =+C17/ =+H$2/ =+C17/ =+H$2/ =+C17/
(0.25*C8 + D8) (H8*25) ((F8 + 0.25*C8)/ (J8*25) ((0.25*C8 + D8)/
(L8*25) ((0.25*C8)/(1 - (N8*25) (1 - E8)) (1 - B8)) B8 + D8)) 9
=+H$2/ =+C18/ =+H$2/ =+C18/ =+H$2/ =+C18/ =+H$2/ =+C18/ (0.25*C9 +
D9) (H9*25) ((F9 + 0.25*C9)/ (J9*25) ((0.25*C9 + D9)/ (L9*25)
((0.25*C9)/(1 - (N9*25) (1 - E9)) (1 - B9)) B9 + D9)) 10 =+H$2/
=+C19/ =+H$2/ =+C19/ =+H$2/ =+C19/ =+H$2/ =+C19/ (0.25*C10 + D10)
(H10*25) ((F10 + 0.25*C10)/ (J10*25) ((0.25*C10 + D10)/ (L10*25)
((0.25*C10)/(1 - (N10*25) (1 - E10)) (1 - B10)) B10 + D10)) 11 12
13 14 =0.016/ 100 15 16 17 18 19 20 21 22 23 24 25 No sorting Units
Remove black Units Select hetero's Units Select hetero's Units
produced produced produced produced 26 with mix with mix and blacks
with mix with mix 27 28 =+H$2/ =+C37/ =+H$2/ =+C37/ =+H$2/ =+C37/
=+H$2/ =+C37/ (0.25*C28 + D28) (H28*25) ((F28 + 0.25*C28)/ (J28*25)
((0.25*C28 + D28)/ (L28*25) ((0.25*C28)/(1 - (N28*25) (1 - E28)) (1
- B28)) B28 + D28)) 29 =+H$2/ =+C38/ =+H$2/ =+C38/ =+H$2/ =+C38/
=+H$2/ =+C38/ (0.25*C29 + D29) (H29*25) ((F29 + 0.25*C29)/ (J29*25)
((0.25*C29 + D29)/ (L29*25) ((0.25*C29)/(1 - (N29*25) (1 - E29)) (1
- B29)) B29 + D29)) 30 =+H$2/ =+C39/ =+H$2/ =+C39/ =+H$2/ =+C39/
=+H$2/ =+C39/ (0.25*C30 + D30) (H30*25) ((F30 + 0.25*C30)/ (J30*25)
((0.25*C30 + D30)/ (L30*25) ((0.25*C30)/(1 - (N30*25) (1 - E30)) (1
- B30)) B30 + D30)) 31 32 33 34 0.00016
Example 5
[0088] In the following example licensing fees are calculated for a
delivery of grain by the grower to the elevator. The bsc level is
set to be 2% in trait fee hectares. The total grain amount is 1000
metric tons. The fraction of bsc in the delivery to the elevator is
0.8%. The current commodity price for standard soybean is
$100/metric ton. The trait fee is assessed as a 2.5% premium over
commodity price on the grain containing the proprietary trait.
Thus, licensing fees may be assessed in the following manner:
First, the elevator determines whether the grain shipment contains
the proprietary trait based on the presence of bsc in the grain.
Then the elevator performs the following set of calculations to
determine the trait fee:
The fraction of grain with the proprietary trait = 0.8 % / 2.0 % =
0.4 ##EQU00001## The grain amount with the proprietary trait = 1000
metric tons .times. 0.4 = 400 metric tons ##EQU00001.2## The trait
fee = ( grain with trait ) .times. ( commodity price ) .times. (
trait fee premium % ) = ( 400 metric tons ) .times. ( $100 / metric
ton ) .times. ( 2.5 % ) = $1 , 000 ##EQU00001.3##
[0089] In view of the above, one of skill in the art should
recognize that the grower is only paying a proprietary trait fee
based on the amount of proprietary grain present.
Example 6
[0090] A method for using mechanical mixing to generate levels of
heterozygous bsc seeds that will deter seed sorting by growers is
as follows: [0091] Step #1 Plant a known quantity of mixed seed
which is homozygous yellow and homozygous black of the same
maturity over a significant area, 10 acres or more, at a high
density in which the plants are crowded within a row. [0092] Step
#2 Harvest all seed produced from this mixed planting. 0.5 to 1% of
the yellow seed harvested should be heterozygous for the "I" gene
(Ii) and thus still yellow, but with a little more black or gray
color, especially in and/or around the hilum. [0093] Step #3 The
harvested seed will be replanted and F1 plants selected based on
the presence of a dominant trait (leaf morphology, or a second
herbicide resistance). [0094] Step #4 The heterozygous yellow seed
will be harvested and increased for a further generation. [0095]
Step #5 The seed increased and sorted as a result of the steps
above would be mixed into varietal seed containing a proprietary
trait.
[0096] In view of the above, one of skill in the art should
recognize that seed sorting of the mixture in Step 5 will be very
difficult for the grower to accomplish.
Example 7
[0097] A method for using mechanical mixing to generate levels of
heterozygous bsc seeds that will deter seed sorting is as follows:
[0098] Step #1 Plant a known quantity of mixed seed which is
homozygous yellow and homozygous black of the same
approximate-maturity over a significant area (e.g., 10 acres or
more) at a high density in which the plants are crowded within a
row. [0099] Step #2 Harvest all seed produced from this mixed
planting. 0.5 to 1% of the yellow seed harvested should be
heterozygous for the "I" gene (Ii) but all seed from the yellow
seeded lines will be yellow, and all seed from the black seeded
lines will be black, due to the maternal control of seed coat
color. [0100] Step #3 All harvested seed would be replanted. [0101]
Step #4 Heterozygous yellow seed will be identified either through
the use of a dominant plant phenotype (leaf shape, or second
herbicide resistance), or through the harvest of seed that exhibits
a slight coloring due to the heterozygous nature of the parental
plants. These F2 seeds would be replanted and increased for a
further generation. [0102] Step #5 The seed increased and sorted as
a result of the steps above would be mixed with bsc seed that is
simply increased and varietal yellow seed containing a proprietary
trait.
Alternatives:
[0103] The homozygous yellow seeds would not need to be sorted out
from the increases following the first initial sorting. The mixture
level would be adjusted to compensate for the presence of this
seed. This does not affect the amount of black or heterozygous seed
sold to or harvested by the grower.
[0104] In view of the above, one of skill in the art should
recognize that seed sorting of the mixture in Step 5 will be very
difficult for the grower to accomplish.
Example 8
[0105] A large scale method for generating hybrid soybean seed is
as follows: [0106] Step #1 Herbicide resistant, black seed coat
seeds are mixed in a 50/50 seed mixture with non-herbicide
resistant yellow seed coat seeds, and randomly planted on a field
large enough to produce at least a hectare of F1 seed. There should
be approximately 1% outcrossing. [0107] Step #2 The field is bulk
harvested and the seeds are replanted in the field at double the
seeding rate. The resulting outcrossed seeds are replanted with the
selfed seed. [0108] Step #3 The field is sprayed with the herbicide
for which herbicide resistance existed in the parental material.
This will eliminate all of the selfed yellow conventional seed.
[0109] Step #4 The remaining seed is harvested. The F2 seed is
separated from the selfed black, herbicide resistant seed through
the use of a color sorter. The yellow seed is retained. [0110] Step
#5 The yellow seed from step 4 (F2 seed) is increased and then used
to mix with seeds containing proprietary traits as a segregating
phenotypic marker.
[0111] In view of the above, one of skill in the art should
recognize that seed sorting of the mixture in Step 5 will be very
difficult for the grower to accomplish.
Example 9
[0112] The following Tables 10 and 11 show a model for a large
scale method for generating hybrid soybean seed described in
Example 8. Table 10 contains the results and Table 11 shows the
appropriate formulas:
TABLE-US-00010 TABLE 10 A B C D E 1 2 Inputs 3 Field size (Ha) 10 4
Planting rate 300000 (seeds/Ha) 5 Outcrossing rate (%) 1% 6
Increase rate (X) 40 7 Mixing rate (%) 2% 8 9 Seeds Kg Units Mixed
units 10 F1 seed produced 1200000 180 7.2 360 11 F2 seed produced
36000000 5400 216 10800 12 F3 seed produced 1200000000 180000 7200
360000
TABLE-US-00011 TABLE 11 A B C D E 1 2 Inputs 3 Field size (Ha) 10 4
Planting rate 300000 (seeds/Ha) 5 Outcrossing rate (%) 0.01 6
Increase rate (X) 40 7 Mixing rate (%) 0.02 8 9 Seeds Kg Units
Mixed units 10 F1 seed produced =+B3*B4*B5*B6 =+B10*0.00015
=+C10/25 =+D10/B$7 11 F2 seed produced =+B10*B6*0.75 =+B11*0.00015
=+C11/25 =+D11/B$7 12 F3 seed produced =+(2/3*B11*B6*0.75) +
=+B12*0.00015 =+C12/25 =+D12/B$7 (1/3*B11*B6) Note: The weight of 1
seed is 0.00015 kg. 1 unit = 25 Kg
Example 10
[0113] The following large scale method for generating hybrid
soybean seed utilizes the model in Example 9 above:
TABLE-US-00012 Step #1 Hybrid Production nursery Size: 10 Ha
Planting rate: 300,000 seeds/Ha Roundup .RTM. spray: No Harvest
all: 300,000 .times. 5 .times. 40 = 360 units Step #2 Select hybrid
progeny (grow F1 plants and harvest F2 seed) Size: 120 Ha Planting
rate: 300,000 seeds/Ha Roundup .RTM. spray: Early Harvest all
remaining: 7,272 units Step #3 Select F2 seed and recycle black RR
seed Seed selected as yellow/RR = 144 units Step #4 Final
production of product for mixing (grow F2 plants) Size: 240 Ha
Planting rate: 100,000 seeds/Ha Roundup .RTM. spray: Early Harvest
all: 4,320 units Step #5 Mechanical mixing Mix product of Step # 4
with commercial seed at a 2% rate.
[0114] When farmers plant the mix generated by Step #5, the
resulting harvest will contain approximately 0.63% black seed.
REFERENCES
[0115] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by reference.
[0116] Nagai, I. 1921. A gentico-physiological study on the
formation of anthocyanin and brown pigments in plants. Tokyo Univ.
Coll. of Agr. J. 8:1-92 [0117] Woodworth C. M., 1921. Inheritance
of cotyledon, seed coat, hilum, and pubescence colors in soy-beans.
Genetics 6:487-553. [0118] Williams L. F. 1952. The inheritance of
certain black and brown pigments in the soy-bean. Genetics 37:
208-215. [0119] Nagai I. and S. Saite. 1923. Linked Factors in
Soybeans. Jap. J. Bot. 1:-121-136. [0120] Williams L. F. 1958.
Alteration of dominance and apparent change indirection of gene
action by a mutation at another locus affecting the pigmentation of
the seed coat of the soybean (Abs.) Tenth Int. Cong. Genet. Proc.
2:315-316. [0121] Williams L. F. 1945. Off-colored seeds in the
Lincoln soybean. Soybean Digest 5(11) 51-61. [0122] International
Rules for Seed Testing 1999 (Seed Science and Technology, Vol. 27,
Supplement, 1999) International Seed Testing Organization [0123]
Van Deynze A. and Pauls, Peter. 1994. "The inheritance of seed
color and vernalization requirement in Brassica napus using doubled
haploid populations," Euphytica 74:77-83.
* * * * *