U.S. patent application number 15/923484 was filed with the patent office on 2018-07-19 for system for imaging and orienting seeds and method of use.
This patent application is currently assigned to Dow AgroSciences LLC. The applicant listed for this patent is Dow AgroSciences LLC. Invention is credited to Sivarama Reddy Chennareddy, Toby Cicak, David T. Gillespie, Donald L. McCarty, II, Rodrigo Sarria.
Application Number | 20180199504 15/923484 |
Document ID | / |
Family ID | 54367025 |
Filed Date | 2018-07-19 |
United States Patent
Application |
20180199504 |
Kind Code |
A1 |
McCarty, II; Donald L. ; et
al. |
July 19, 2018 |
SYSTEM FOR IMAGING AND ORIENTING SEEDS AND METHOD OF USE
Abstract
A system and method for the automated or semi-automated
imagining and orienting of seeds to prepare the seeds for
transformation and transgenic engineering.
Inventors: |
McCarty, II; Donald L.;
(Freeland, MI) ; Chennareddy; Sivarama Reddy;
(West Lafayette, IN) ; Cicak; Toby; (Indianapolis,
IN) ; Sarria; Rodrigo; (West Lafayette, IN) ;
Gillespie; David T.; (Pearland, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dow AgroSciences LLC |
Indianapolis |
IN |
US |
|
|
Assignee: |
Dow AgroSciences LLC
Indianapolis
IN
|
Family ID: |
54367025 |
Appl. No.: |
15/923484 |
Filed: |
March 16, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14704691 |
May 5, 2015 |
9924626 |
|
|
15923484 |
|
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|
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61989266 |
May 6, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G06T 1/0007 20130101;
G05B 2219/39107 20130101; G01N 35/0099 20130101; G06T 7/70
20170101; G06T 7/73 20170101; G01N 1/286 20130101; G05B 2219/39106
20130101; B26D 5/007 20130101; G06T 2207/30172 20130101; G06T 7/49
20170101; G06T 2207/30004 20130101; G05B 2219/37555 20130101; G06T
2207/20068 20130101; B25J 9/1697 20130101; G05B 2219/37608
20130101; G06T 2207/10004 20130101; A01C 1/00 20130101; G06T 7/0002
20130101; G06T 7/12 20170101 |
International
Class: |
A01C 1/00 20060101
A01C001/00; G06T 7/49 20170101 G06T007/49; G06T 7/73 20170101
G06T007/73; G06T 7/70 20170101 G06T007/70; B25J 9/16 20060101
B25J009/16; G06T 1/00 20060101 G06T001/00; G06T 7/00 20170101
G06T007/00; G06T 7/12 20170101 G06T007/12 |
Claims
1. A method for imaging a seed, the method comprising: using a
robotic arm to position a seed for imaging, capturing a plurality
of images of the seed, identifying a location of the embryo of the
seed based on the plurality of captured images, identifying an edge
of the hilum nearest the identified location based on the plurality
of captured images, identifying a point between the edge of the
hilum and the outer edge of the seed at which to trim an embryonic
axis of the seed, and moving the seed with the robotic arm to
orient the seed in a position based identified point at which to
trim the embryonic axis of the seed.
2. The method of claim 1, wherein identifying the location of the
embryo of the seed includes analyzing the captured images to locate
the embryonic axis of the seed.
3. The method of claim 2, wherein identifying the point between the
edge of the hilum and the outer edge of the seed at which to trim
the embryonic axis of the seed includes identifying the edge of the
hilum nearest the embryonic axis.
4. The method of claim 3, wherein identifying the point between the
edge of the hilum and the outer edge of the seed at which to trim
the embryonic axis of the seed includes identifying the outer edge
of the seed on the same side of the seed as the embryonic axis.
5. The method of claim 1, wherein using the robotic arm to position
the seed for imaging includes engaging the seed with a grip of the
robotic arm.
6. The method of claim 5, further comprising calculating a distance
between the identified point at which to trim the embryonic axis of
the seed and a center point of the grip.
7. The method of claim 5, wherein engaging the seed with the grip
of the robotic arm includes attaching the seed to the grip via a
suction force.
8. A method for imaging a seed, the method comprising: using a
robotic arm to position a hydrated seed for imaging, capturing a
plurality of images of the hydrated seed, determining an
orientation of the hydrated seed and a location of the hilum of the
hydrated seed based on the plurality of captured images, and moving
the hydrated seed with a robotic arm to orient the hydrated seed in
a position for cutting the seed based on the determined orientation
of the hydrated seed and the location of the hilum.
9. The method of claim 8, further comprising analyzing the captured
images to locate an embryonic axis of the seed.
10. The method of claim 9, wherein determining the orientation of
the hydrated seed and the location of the hilum of the hydrated
seed includes identifying an edge of the hilum nearest the
embryonic axis.
11. The method of claim 10, wherein determining the orientation of
the hydrated seed and the location of the hilum of the hydrated
seed includes identifying the outer edge of the seed on the same
side of the seed as the embryonic axis.
12. The method of claim 9, wherein using the robotic arm to
position the hydrated seed for imaging includes engaging the seed
with a grip of the robotic arm.
13. The method of claim 12, wherein engaging the seed with the grip
of the robotic arm includes attaching the seed to the grip via a
suction force.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/704,691, now U.S. Pat. No. 9,924,626, filed
on May 5, 2015, which claims priority to U.S. Provisional Patent
Application Ser. No. 61/989,266, filed on May 6, 2014, the entire
disclosures of both of which are expressly incorporated herein by
reference.
CROSS-REFERENCE TO RELATED U.S. PATENT APPLICATION
[0002] Cross-reference is made to U.S. Provisional Patent
Application Ser. No. 61/989,275 entitled "SYSTEM FOR SEED
PREPARATION AND METHOD OF USE" by Donald L. McCarty, II et al.,
which was filed on May 6, 2014; and to U.S. Provisional Patent
Application Ser. No. 61/989,276 entitled "SYSTEM FOR CUTTING AND
PREPARING SEEDS AND METHOD OF USE" by Donald L. McCarty, II et al.,
which was filed on May 6, 2014, each of which is expressly
incorporated herein by reference.
TECHNICAL FIELD
[0003] The present disclosure relates generally to devices for
preparing seeds for use in plant breeding, and, more specifically,
to a device for preparing seeds and seed explants for gene
transformation and transgenic engineering.
BACKGROUND
[0004] Soybean (Glycine max) is one of the most important
agricultural crops, with an annual crop yield of more than 200
million metric tons, and an estimated value exceeding 40 billion
U.S. dollars worldwide. Soybean accounts for over 97% of all
oilseed production globally. Thus, reliable and efficient methods
for improving the quality and yield of this valuable crop are of
significant interest.
[0005] Traditional breeding methods for improving soybean have been
constrained because the majority of soybean cultivars are derived
from only a few parental lines, leading to a narrow germplasm base
for breeding. Christou et al., TIBTECH 8:145-151 (1990). Modern
research efforts have focused on plant genetic engineering
techniques to improve soybean production. Transgenic methods are
designed to introduce desired genes into the heritable germline of
crop plants to generate elite plant lines. The approach has
successfully increased the resistance of several other crop plants
to disease, insects, and herbicides, while improving nutritional
value.
[0006] Several methods have been developed for transferring genes
into plant tissue, including high velocity microprojection,
microinjection, electroporation, and direct DNA uptake.
Agrobacterium-mediated gene transformation has more recently been
used to introduce genes of interest into soybeans. However,
soybeans have proven to be a challenging system for transgenic
engineering. Efficient transformation and regeneration of soybean
explants is difficult to achieve, and frequently hard to
repeat.
[0007] Agrobacterium tumefaciens, a pathogenic, soil-dwelling
bacterium, has the inherent ability to transfer its DNA, called
T-DNA, into host plant cells and to induce the host cells to
produce metabolites useful for bacterial nutrition. Using
recombinant techniques, some or all of the T-DNA may be replaced
with a gene or genes of interest, creating a bacterial vector
useful for transforming the host plant. Agrobacterium-mediated gene
transfer is typically directed at undifferentiated cells in tissue
culture, but may also be directed at differentiated cells taken
from the leaf or stem of the plant. A number of procedures have
been developed for Agrobacterium-mediated transformation of
soybean, which may loosely be classified based on the explant
tissue subjected to transformation.
[0008] U.S. Pat. No. 7,696,408, Olhoft, et al., discloses a
cotyledonary node method for transforming both monocotyledonous and
dicotyledonous plants. The "cot node" method involves removing the
hypocotyl from 5-7 day old soybean seedlings by cutting just below
the cotyledonary node, splitting and separating the remaining
hypocotyl segment with the cotyledons, and removing the epicotyl
from the cotyledon. The cotyledonary explant is wounded in the
region of the axillary bud and/or cotyledonary node, and cultivated
with Agrobacterium tumefaciens for five days in the dark. The
method requires in-vitro germination of the seeds, and the wounding
step introduces significant variability.
[0009] U.S. Pat. No. 6,384,301, Martinelli et al., discloses
Agrobacterium-mediated gene delivery into living meristem tissue
from soybean embryos excised from soybean seeds, followed by
culturing of the meristem explant with a selection agent and
hormone to induce shoot formation. Like the "cot node" method, the
meristem explants are preferably wounded prior to infection.
[0010] U.S. Pat. No. 7,473,822, Paz et al., discloses a modified
cotyledonary node method called the "half-seed explant" method.
Mature soybean seeds are imbibed, surface-sterilized and split
along the hilum. Prior to infection, the embryonic axis and shoots
are completely removed, but no other wounding occurs.
Agrobacterium-mediated transformation proceeds, potential
transformants are selected, and explants are regenerated on
selection medium.
[0011] Transformation efficiencies remain relatively low with these
methods, on the order of 0.3% to 2.8% for the "cot node" method,
1.2 to 4.7% for the "meristem explant" method, and between 3.2% and
8.7% (overall 4.9%) for the "half-seed explant" method.
Transformation efficiencies of approximately 3% are typical in the
art.
[0012] An improved "split-seed" transgenic protocol may accelerate
future production and development of transgenic soybean products.
An efficient and high-throughput method for stable integration of a
transgene into soybean tissue would facilitate breeding programs
and have the potential to increase crop productivity.
SUMMARY
[0013] A method and apparatus for automated seed preparation is
disclosed. According to one aspect, the method includes locating
the seed on a surface or container, engaging the seed with an
automated tool, orienting the seed for cutting or wounding, and
cutting or wounding the seed when the seed is oriented. The method
may also include partially cutting the embryonic axis of the seed.
In some embodiments, the cut or wounded seed is transformed with
exogenous DNA.
[0014] The scope of the disclosure is not limited to the specified
structures or the specific terms used. For example, the term
"robotic arm" may be substituted with the term "automated tool."
Additionally, the terms "surface" or "container" may be substituted
for the term "tray," and the term "cutting block" may be
substituted with the terms "cutting surface," "support block," or
"block."
[0015] The automated seed preparation method may include capturing
an image of a tray including at least one seed, locating the seed
on the surface or container (e.g., the tray) based on a captured
image, gripping the seed with an automated tool (e.g., the robotic
arm), orienting the seed on a cutting surface (e.g., the cutting
block) for bisection of the seed, and bisecting the seed when the
seed is oriented on the cutting surface. In some embodiments, the
method may also include partially cutting the embryonic axis of the
seed. In some embodiments, locating the seed may comprise locating
the seed on a tray having a plurality of seeds placed thereon.
[0016] In some embodiments, the method may further comprise
operating the robotic arm to move the seed from the tray to a
separate location, capturing a plurality of images of the seed in
the separate location, and determining a proper orientation of the
seed for bisection based on the plurality of captured images. In
some embodiments, the plurality of images may be captured by one
camera that captures an image set from one or more perspectives. In
other embodiments, capturing the plurality of images may comprise
operating a first camera to capture a first image set of the seed
from a first perspective, and operating a second camera to capture
a second image set of the seed from a second perspective different
from the first perspective. As used herein, an image set may
include one image or a plurality of images.
[0017] Additionally, in some embodiments, determining the proper
orientation of the seed may comprise locating a center of a hilum
of the seed and a longitudinal axis of the seed.
[0018] In some embodiments, orienting the seed on the cutting
surface (e.g., the cutting block) for bisection of the seed may
comprise aligning the seed with a cutting blade of a cutting device
along an imaginary plane defined by the center of the hilum of the
seed and the longitudinal axis of the seed.
[0019] In some embodiments, the method may further comprise
trimming an embryonic axis of the seed when the seed is located on
the cutting surface. In some embodiments, determining the proper
orientation of the seed for bisection based on the plurality of
captured images may also include determining the proper orientation
of the seed for trimming the embryonic axis. Trimming the embryonic
axis of the seed may include positioning a cutting blade
perpendicular to a longitudinal axis of the seed.
[0020] In some embodiments, bisecting the seed on the cutting
surface may comprise bisecting the seed after trimming the
embryonic axis of the seed. Additionally, in some embodiments,
bisecting the seed on the cutting surface may comprise cutting
through less than an entirety of the seed. In some embodiments, the
method may further comprise moving the bisected seed to an
Agrobacterium tumefaciens solution.
[0021] In some embodiments, the method may comprise sterilizing a
grip of the automated tool (e.g., the robotic arm) prior to
gripping the seed. The method may comprise operating the automated
tool or robotic arm to select a cutting blade, and positioning the
cutting blade on a cutting device. The method may further comprise
bisecting the seed when the seed is oriented on the cutting block
by inserting the cutting blade into the seed. In some embodiments,
the method may include gripping the cutting blade with the same or
a different automated tool after bisecting the seed and operating
the automated tool to replace the cutting blade with a second
cutting blade on the cutting device.
[0022] According to another aspect, a seed preparation apparatus
comprises a first camera configured to capture a first image set of
a seed placed on a surface or a tray, a robotic arm operable to
grip the seed and to move the seed from the surface or the tray to
a lighted chamber, a second camera configured to capture a second
image set of the seed within the lighted chamber, and a cutting
block configured to receive the seed. The robotic arm is further
operable to position the seed on the cutting block in a proper
orientation for bisection of the seed.
[0023] In some embodiments, the seed preparation apparatus may
comprise a light source positioned on a first side of the tray to
illuminate the seeds on the tray. In some embodiments, the lighted
chamber may be defined in a lighted dome.
[0024] In some embodiments, the seed preparation apparatus may
comprise a third camera configured to capture a third image set of
the seed within the lighted chamber, and an electronic controller
configured to analyze the second image set and the third image set
to determine the proper orientation of the seed.
[0025] In some embodiments, the seed preparation apparatus may
comprise a light source configured to light an interior of the
lighted chamber.
[0026] In some embodiments, the electronic controller may be
further configured to analyze the first image set to locate the
seed on the tray.
[0027] According to another aspect, a seed preparation apparatus
comprises a chamber, a first camera configured to capture a first
image set of a seed on a tray, a second camera configured to
capture a second image set of the seed within the chamber, a
cutting device configured to bisect the seed, a robotic arm
including a gripping device to grip the seed for movement, and an
electronic controller. The electronic controller is configured to
locate the seed on the tray based on the first image set, operate
the robotic arm to grasp the seed on the tray and move the seed to
the cutting device in an orientation based on the second image set,
and operate the cutting device to bisect the seed.
[0028] In some embodiments, the electronic controller may be
configured to operate the robotic arm to move the seed from the
tray to the chamber, and operate the second camera to capture the
second image set.
[0029] In some embodiments, the electronic controller may be
configured to analyze a plurality of images of the seed to
determine a proper orientation of the seed for bisection of the
seed and trimming an embryonic axis of the seed.
[0030] In some embodiments, the robotic arm may be configured to
move the seed to the cutting device to position the seed in the
proper orientation, and the cutting device is configured to trim
the embryonic axis of the seed while the seed is positioned in the
cutting device in the proper orientation.
[0031] According to another aspect of the disclosure, a cutting
block is disclosed. The cutting block comprises a body including a
front wall and a substantially planar upper wall extending away
from the front wall. A first opening is defined in the front wall,
a second opening is defined in the upper wall, and a plurality of
inner walls extend inwardly from the first opening and the second
opening to define a slot in the front wall and the upper wall. The
slot is sized to receive a cutting tool. The cutting block is sized
to support a seed such as a soybean seed or any seed of that size
that cutting tool may be advanced along the slot into contact with
the seed.
[0032] In some embodiments, the upper wall may extend from the
front wall to a rear edge. The body may further include a
substantially planar side wall extending upwardly from the rear
edge. In some embodiments, the side wall may be a first side wall
extending from the rear edge of the upper wall to an upper edge The
body may further include a second side wall extending from the
upper edge of the first side wall. The second side wall may extend
obliquely relative to the first side wall and the upper wall of the
body.
[0033] In some embodiments, the second side wall may extend from
the upper edge of the first side wall to a top edge, and the body
may further include a top wall extending from the top edge of the
second side wall. The top wall may extend obliquely relative to the
second side wall.
[0034] In some embodiments, the top wall may extend parallel to the
upper wall of the cutting block.
[0035] In some embodiments, the slot may extend from the first
opening in the front wall to a back edge positioned between the
front wall and the rear edge of the upper wall.
[0036] In some embodiments, the first opening may be positioned in
a center of the front wall. In some embodiments, the body may be
formed as a single monolithic metallic body. In some embodiments,
the body may be secured to a surface with an automated cutting
system.
[0037] In further embodiments, a combination is disclosed. The
combination includes each cutting block herein with a seed such as
a soybean seed o any seed of that size. The soybean seed may be
cut, bisected, trimmed, or otherwise wounded for transformation. In
some embodiments, the embryonic axis of the seed may be trimmed for
transformation.
[0038] According to another aspect, a cutting system is disclosed.
The cutting system includes an automated cutting system including a
cutting tool, and a cutting block including an upper wall and a
slot defined in the upper wall that is sized to receive the cutting
tool of the automated cutting system. The automated cutting system
is operable to move the cutting tool linearly along a first axis
relative to the cutting block, and rotate the cutting tool about
the first axis to position the cutting tool for insertion into the
slot.
[0039] In some embodiments, the automated cutting system may
further include an electric motor operable to move the cutting tool
linearly along the first axis, and a pneumatic device operable to
rotate the cutting tool about the first axis.
[0040] In some embodiments, the automated cutting system further
may include a pair of movable jaws configured to receive the
cutting tool. The pair of movable jaws may be operable to move
between an unlocked position in which the cutting tool is removable
from the jaws, and a locked position in which the cutting tool is
retained on the jaws.
[0041] In some embodiments, the automated cutting system further
may include a second pneumatic device operable to move the pair of
jaws between the unlocked position and the locked position.
[0042] In some embodiments, the automated cutting system further
may include an electronic controller including a processor, a
memory device, and a plurality of instructions stored in the memory
device, which, when executed by the processor, cause the processor
to operate a first compressed air source to move the pair of jaws
from the unlocked position to the locked position, operate a second
compressed air source to rotate the cutting tool about the first
axis to an orientation in which the cutting tool extends
vertically, and operate the first electric motor to advance the
cutting tool into the slot defined in the cutting block. In some
embodiments, the electronic controller may further include a
plurality of instruction, which, when executed by the processor,
cause the processor to operate the first electric motor to remove
the cutting tool from the slot defined in the cutting block,
operate the second compressed air source to rotate the cutting tool
about the first axis to a second orientation in which the cutting
tool extends horizontally, and operate the first electric motor to
advance the cutting tool over the upper wall of the cutting
block.
[0043] In some embodiments, the cutting block may include a front
wall and the substantially planar upper wall extends away from the
front wall, and a first opening is defined in the front wall, a
second opening is defined in the upper wall, and a plurality of
inner walls extend inwardly from the first opening and the second
opening to define the slot in the front wall and the upper
wall.
[0044] In some embodiments, the cutting tool may be removably
coupled to the automated cutting system.
[0045] According to another aspect, a method of cutting a seed is
disclosed. The method includes advancing a cutting tool along a
first axis into a slot defined in a cutting block and to make a
first cut in the seed, rotating the cutting tool about the first
axis, and advancing the cutting tool into the seed to make a second
cut. In some embodiments, the cutting tool may be rotated by
operating a compressed air source or an electric motor. In some
embodiments, the cutting tool may be advanced into the seed by
operating one or more electric motors.
[0046] In some embodiments, the method may comprise positioning the
cutting tool on a pair of jaws, and moving the pair of jaws to
secure the cutting tool to the pair of jaws. In some embodiments,
the pair of jaws may be moved apart to engage the cutting tool.
Additionally, in some embodiments, the pair of jaws may be moved by
operating a compressed air source.
[0047] In some embodiments, positioning the cutting tool on the
pair of jaws may include attaching the cutting tool to an automated
tool such as a robotic arm.
[0048] In some embodiments, the method may further comprise
operating a negative pressure source to attach the cutting tool to
the robotic arm via suction.
[0049] According to another aspect, a method for imaging a seed is
disclosed. The method includes using an automated tool such as a
robotic arm to position a seed including a hilum within a lighted
structure such as a dome, projecting the seed onto a first plane
extending perpendicular to a center axis of the lighted dome,
rotating the seed to orient the seed parallel to a first imaginary
horizontal line positioned in the first plane, projecting the seed
onto a second plane extending perpendicular to the first plane,
orienting the seed parallel to a second imaginary horizontal line
positioned in the second plane, identifying a distance between the
hilum of the seed and the second imaginary horizontal line, and
orienting the seed to position the hilum on the second imaginary
horizontal line based on the identified distance.
[0050] As described above, the scope of the disclosure is not
limited to the disclosed structures or terms used. Thus, the term
"lighted dome" may be substituted with, for example, the term
"lighted structure."
[0051] In some embodiments, orienting the seed to position the
hilum on the second imaging horizontal line may comprise orienting
the seed to position a center of the hilum on the second imaginary
horizontal line. In some embodiments, orienting the seed to
position the center of the hilum on the second imaginary horizontal
line may comprise orienting the seed such that a center of mass of
the hilum is coincident with a center of mass of the seed.
[0052] Additionally, in some embodiments, the method may further
comprise identifying a location of the embryo of the seed,
identifying an edge of the hilum nearest the identified location
and an outer edge of the seed along the second imaginary horizontal
line, and identifying a point between the edge of the hilum and the
outer edge of the seed at which to trim an embryonic axis of the
seed. In some embodiments, identifying the location of the embryo
may comprise analyzing one or more projections of the seed onto the
second plane using feature matching.
[0053] In some embodiments, projecting the seed onto the first
plane may comprise capturing a first image set with a first camera,
and projecting the seed on to a second plane may comprise capturing
a second image set with a second camera.
[0054] In some embodiments, the first camera may have an optical
axis parallel to an optical axis of the second camera, and
capturing the first image set with the first camera may comprise
capturing light reflected off a mirror extending at a forty-five
degree angle relative to the optical axis of the first camera.
[0055] In some embodiments, rotating the seed to orient the seed
parallel to the first imaginary horizontal line may comprise
rotating the seed in response to determining the seed is not
oriented parallel to the first imaginary horizontal line. In some
embodiments, orienting the seed parallel to the second imaginary
horizontal line may comprise orienting the seed in response to
determining the seed is not oriented parallel to the second
imaginary horizontal line, and orienting the seed to position the
hilum on the secondary imaginary horizontal line may comprise
orienting the seed in response to determining the hilum is not
positioned on the second imaginary horizontal line.
[0056] In some embodiments, the method may further comprise
analyzing a first image set corresponding with the projection of
the seed onto the first plane to determine an orientation of the
seed relative to the first imaginary horizontal line, and analyzing
a second image set corresponding with the projection of the seed
onto the second plane to determine an orientation of the seed
relative to the second imaginary horizontal line.
[0057] In some embodiments, analyzing the second image set may
comprise identifying a first longitudinal end and a second
longitudinal end of the seed, identifying a left rectangular
vertical cross section of the seed at the first longitudinal end,
identifying a right rectangular vertical cross section of the seed
at the second longitudinal end, determining a center of mass of
each of the left rectangular vertical cross section and the right
rectangular cross section, and interconnecting the centers of mass
of the left rectangular vertical cross section and the right
rectangular cross section with an imaginary line segment. In some
embodiments, orienting the seed parallel to the second imaginary
horizontal line may comprise orienting the seed such that the line
segment is parallel to the second imaginary horizontal line.
[0058] In some embodiments, each of the left rectangular vertical
cross section and the right rectangular vertical cross section may
have a horizontal width equal to at least ten image pixels.
[0059] In some embodiments, analyzing the second image set may
further comprise determining an angle of the line segment relative
to the second imaginary horizontal line, and an amount of rotation
of the seed to orient the line segment parallel to the second
imaginary horizontal line is based on the determined angle.
[0060] In some embodiments, the method may further comprise
projecting the seed onto the second plane in response to orienting
the seed parallel to the second imaginary horizontal line, and
analyzing a third image set corresponding with the projection of
the seed onto the second plane in response to orienting the seed
parallel to the second imaginary horizontal line to identify the
distance between the hilum and the second imaginary horizontal
line.
[0061] In some embodiments, analyzing the third image set may
comprise identifying a longitudinal end of the seed, determining a
center of mass of each of the longitudinal end and the hilum, and
interconnecting the centers of mass of the longitudinal end and the
hilum with an imaginary line segment. Additionally, in some
embodiments, orienting the seed to position the hilum on the second
imaginary horizontal line may comprise orienting the seed such that
the line segment is coincident with the second imaginary horizontal
line.
[0062] In some embodiments, analyzing the third image set may
further comprise determining an angle of the line segment relative
to the second imaginary horizontal line, and an amount of movement
of the seed to orient the line segment coincident with the second
imaginary horizontal line is based on the determined angle.
[0063] In some embodiments, the method may further comprise
determining a height of the seed for positioning a cutting blade
based on the projection of the seed onto the second plane. The
height may be a width of the seed in a direction perpendicular to
the second imaginary horizontal line. In some embodiments, the
method may further include attaching the seed to the robotic arm
via a suction force.
[0064] According to another aspect, a method for imaging a seed
includes capturing a plurality of images of a seed, determining an
orientation of the seed and a location of the hilum of the seed
based on the plurality of captured images, and moving the seed with
a robotic arm to orient the seed in a position based on the
determined orientation of the seed and the location of the
hilum.
[0065] In some embodiments, capturing the plurality of images may
comprise capturing a first image set of the seed with a first
camera from a first perspective, and capturing a second image set
of the seed with a second camera from a second perspective
perpendicular to the first perspective.
[0066] In some embodiments, determining the orientation of the seed
may comprise determining an orientation of the seed relative to a
first border line of the first captured image set, and determining
an orientation of the seed relative to a second border line of the
second captured image set.
[0067] According to another aspect, a seed imaging apparatus
includes a robotic arm, one or more light sources, a hollow body
having a center axis and configured to be lighted by the one or
more light sources, a first camera configured to capture a first
image set of a seed positioned within the hollow body. The first
image set is captured from a first perspective along the center
axis. The seed imagining apparatus includes a second camera
configured to capture a second image set of the seed from a second
perspective along a second axis perpendicular to the center axis,
and an electronic controller configured to analyze the first image
set and the second image set to determine a proper orientation of
the seed for bisection and to instruct the robotic arm to move the
seed into the proper orientation.
[0068] In some embodiments, an optical axis of the first camera may
be parallel to an optical axis of the second camera, and the first
camera may be configured to capture light reflected off a mirror
that extends at a forty-five degree angle relative to the optical
axis of the first camera.
[0069] In some embodiments, the robotic arm may be configured to
secure the seed by applying a suction force to a side of the
seed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0070] The detailed description particularly refers to the
following figures, in which:
[0071] FIG. 1 is a perspective view of a system for preparing seeds
for gene transformation;
[0072] FIG. 2 is a top plan view of the system of FIG. 1;
[0073] FIG. 3 is an exploded perspective view of a portion of a
dock of the system of FIG. 2;
[0074] FIG. 4 is a perspective view of an imaging station of the
system of FIG. 2;
[0075] FIG. 5 is an exploded perspective view of the imaging
station of FIG. 4;
[0076] FIG. 6 is a perspective view of a cutting device of the
system of FIG. 2;
[0077] FIG. 7 is an exploded perspective view of a cutting block of
the cutting device of FIG. 6;
[0078] FIG. 8 is a top plan view of the cutting block of FIG.
7;
[0079] FIG. 9 is a side elevation view of the cutting block of
FIGS. 7-8;
[0080] FIG. 10A is a top plan view of the cutting device of FIG. 6
showing the jaws in a disengaged position;
[0081] FIG. 10B is a front perspective view of the cutting device
of FIG. 6 showing the jaws in a disengaged position;
[0082] FIG. 11A is a view similar to FIG. 10A showing the jaws in
an engaged position;
[0083] FIG. 11B is a view similar to FIG. 10B showing the jaws in
an engaged position;
[0084] FIG. 12 is a perspective view of a cutting tool tray of the
system of FIG. 1;
[0085] FIG. 13 is a perspective view of a grip assembly of a
robotic arm of the system of FIG. 1;
[0086] FIG. 14 is a simplified block diagram of the system of FIG.
1;
[0087] FIGS. 15-16 are block diagrams showing an illustrative
operating procedure for the system of FIG. 1;
[0088] FIGS. 17-19 are block diagrams showing an illustrative
procedure for determining a desired cutting position and cutting
depth for a soybean seed;
[0089] FIGS. 20-26 are illustrations of various preliminary actions
in the operating procedure of FIGS. 15-16, including sterilizing
the grips of the system of FIG. 1 and selecting a cutting tool;
[0090] FIGS. 27-29 are illustrations of an image capture process of
the operating procedure of FIGS. 15-16 to identify a seed to be
picked up by the system of FIG. 1;
[0091] FIGS. 30-31 are illustrations of the system of FIG. 1 moving
a seed to an imaging station of the system;
[0092] FIGS. 32-55 are illustrations of images created during the
procedure of FIGS. 17-19;
[0093] FIGS. 56-59 are illustrations of the system of FIG. 1
cutting a seed to prepare the seed for gene transformation;
[0094] FIG. 60 is a plan view of a soybean seed;
[0095] FIG. 61 is a side elevation view of the soybean seed of FIG.
60;
[0096] FIG. 62 is a cross-sectional elevation view of the soybean
seed taken along the line 62-62 in FIG. 60;
[0097] FIG. 63 is a cross-sectional elevation view of the soybean
seed taken along the line 63-63 in FIG. 57;
[0098] FIG. 64 is a cross-sectional elevation view of the soybean
seed taken along the line 64-64 in FIG. 59; and
[0099] FIG. 65 is a plan view of a pair of cotyledon segments
prepared using the system of FIG. 1.
DETAILED DESCRIPTION OF THE DRAWINGS
[0100] While the concepts of the present disclosure are susceptible
to various modifications and alternative forms, specific exemplary
embodiments thereof have been shown by way of example in the
drawings and will herein be described in detail. It should be
understood, however, that there is no intent to limit the concepts
of the present disclosure to the particular forms disclosed, but on
the contrary, the intention is to cover all modifications,
equivalents, and alternatives falling within the spirit and scope
of the invention as defined by the appended claims.
[0101] As used herein, a "cotyledon" may generally refer to an
embryonic leaf or "primary leaf" of the embryo of a seed plant. A
cotyledon is also referred to in the art as a "seed leaf."
Dicotyledonous species, such as soybean, have two cotyledons. A
cotyledon segment refers to any portion of a cotyledon, whether it
be an entire or whole cotyledon or a fragment or partial portion of
a cotyledon. The "cotyledonary node" refers to the point of
attachment of the cotyledons to the embyro in the seed or seedling,
and may generally refer to the tissue associated with that point of
attachment.
[0102] As used herein, the term "grasping" refers to holding or
seizing the soybean seed with a tool. Any subsequent mechanism or
action that allows the soybean seed to be firmly clasped is
considered within the scope of the term grasping.
[0103] As used herein, the term "cutting blade" refers to any
cutting tool such as a razor, knife, water knife, scalpel, chisel,
cutter, lance and the like suitable for cutting or wounding a seed
for transformation. In the embodiments disclosed herein, each
reference to a cutting blade may be substituted with a laser or
microlaser emission for cutting or wounding a seed for
transformation.
[0104] As used herein, the term "seed coat" refers to an integument
of the ovule that serves as a seed's protective coat. Seed coat may
be described by the alternative descriptive terms of "testa" or
"husk", in addition to other similar terms known in the art. Seed
coats may contain hydrophobic substances such as suberin, cutin,
lignin, callose, pectin, waxes, and insoluble products of phenolic
oxidation. In legumes, like soybean, the testa contains a palisade
layer of thick-walled macrosclereid cells, whose caps extend into a
suberized sub-cuticle, with a waxy cuticle external to the thicker
suberin layer.
[0105] As used herein, the terms "embryonic axis" or "embryo axis"
refer to the major portion of the embryo of the plant, and
generally includes the epicotyl and hypocotyl.
[0106] As used herein, the term "genetically modified" or
"transgenic" plant refers to a plant cell, plant tissue, plant
part, plant germplasm, or plant which comprises a preselected DNA
sequence which is introduced into the genome of a plant cell, plant
tissue, plant part, plant germplasm, or plant by
transformation.
[0107] As used herein, the term "transgenic," "heterologous,"
"introduced," or "foreign" DNA or gene refer to a recombinant DNA
sequence or gene that does not naturally occur in the genome of the
plant that is the recipient of the recombinant DNA or gene, or that
occurs in the recipient plant at a different location or
association in the genome than in the untransformed plant.
[0108] As used herein, the term "explant" refers to a piece of
soybean tissue that is removed or isolated from a donor plant
(e.g., from a donor seed), cultured in vitro, and is capable of
growth in a suitable media.
[0109] As used herein, the term "plant" refers to either a whole
plant, plant tissue, plant part, including pollen, seeds, or an
embryo, plant germplasm, plant cell, or group of plants. The class
of plants that can be used in the method of the invention is not
limited to soybeans, but may generally include any plants that are
amenable to transformation techniques, including both
monocotyledonous and dicotyledonous plants.
[0110] As used herein the term "transformation" refers to the
transfer and integration of a nucleic acid or fragment into a host
organism, resulting in genetically stable inheritance. Host
organisms containing the transformed nucleic acid fragments are
referred to as "transgenic" or "recombinant" or "transformed"
organisms. Known methods of transformation include Agrobacterium
tumefaciens or Agrobacterium rhizogenes mediated transformation,
calcium phosphate transformation, polybrene transformation,
protoplast fusion, electroporation, ultrasonic methods (e.g.,
sonoporation), liposome transformation, microinjection, naked DNA,
plasmid vectors, viral vectors, biolistics (microparticle
bombardment), silicon carbide WHISKERS.TM. mediated transformation,
aerosol beaming, or PEG transformation as well as other possible
methods. Referring to FIG. 1, a system 10 for preparing seeds or
seed explants for gene transformation by any known method is
shown.
[0111] The system 10 is illustratively configured to prepare
soybean seeds (hereinafter seeds 12) as part of a transgenic
protocol and the development of transgenic soybean products.
Exemplary transgenic protocols are described in U.S. patent
application Ser. No. 14/133,370 entitled "IMPROVED SOYBEAN
TRANSFORMATION FOR EFFICIENT AND HIGH-THROUGHPUT TRANSGENIC EVENT
PRODUCTION" and U.S. patent application Ser. No. 14/134,883
entitled "IMPROVED SOYBEAN TRANSFORMATION FOR EFFICIENT AND
HIGH-THROUGHPUT TRANSGENIC EVENT PRODUCTION," which are expressly
incorporated herein by reference. It should be appreciated that any
of the devices and methods described herein can be used in
connection with the transformation methods disclosed in those
applications. It should also be appreciated that in other
embodiments any of the devices and methods described herein may be
configured for use with other classes of plants that are amenable
to transformation techniques, including both monocotyledonous and
dicotyledonous plants.
[0112] The system 10 includes a number of processing stations 14
and a pair of robotic arms 16 that move seeds 12 between the
processing stations 14. In the illustrative embodiment, each
robotic arm 16 is an Epson model C3 six-axis articulated arm that
is configured to operate independently of the other robotic arm. In
other embodiments, the robotic arms 16 may have a different number
of degrees of freedom than those described herein. For example, the
robotic arms 16 may be embodied as robotic arms having at least
independent axes. Each arm 16 includes a grip 18 configured to
grasp and hold a seed 12. The system 10 may be operated with one of
the arms 16 out-of service. It should be appreciated that in other
embodiments the system may include only a single robotic arm 16 to
move the seeds 12 between the processing stations 14. Additionally,
in the illustrative embodiment, each robotic arm 16 is capable of
rotating the corresponding grip 18 about its axis by at least 180
degrees.
[0113] As shown in FIG. 2, the processing stations 14 and the
robotic arms 16 are arranged on a table 20. The processing stations
14 include a dock 22 positioned at the front of the table 20. The
dock 22 includes a pair of delivery areas 24 where seeds 12 may be
positioned for processing by the system 10 and a pair of receiving
areas 26 where seeds 12 may be positioned after processing by the
system 10. The stations 14 also include an imaging station 28 that
is operable to capture a number of images of the seeds 12. The
system 10 also includes a cutting station 30 that is operable to
cut each seed 12 based on the images captured by the station 28.
The system 10 also includes a sterilization device 32 that is
configured to sterilize each grip 18 of the robotic arms 16 and a
bin or tray 34 that receives cutting blades for use the cutting
station 30.
[0114] In use, the system 10 may be operated to cut automatically a
number of soybean seeds 12 for transformation. To do so, the system
10 may locate one of the seeds 12 on a plate 36 positioned on one
of the delivery areas 24 of the dock 22. The system 10 may then
operate the robotic arm 16 closest to the plate 36 to grasp the
selected seed 12 with the grip 18 and move the seed 12 to the
imaging station 28. After a series of images of the seed 12 are
taken, the arm 16 may advance the seed 12 to the cutting station 30
such that one or more cuts may be made to the seed 12 to prepare it
for transformation. After the seed 12 is cut, the arm 16 may move
the seed 12 to another plate 38 positioned on one of the receiving
areas 26 of the dock 22. A user may then remove the plate 38
including the cut seed to further process the seed in accordance
with the transgenic protocol. Each of these processing steps and
the various components of the system 10 are described in greater
detail below in reference to FIGS. 3-59.
[0115] Referring now to FIG. 3, a portion of the dock 22 and one of
the delivery areas 24 is shown in greater detail. In the
illustrative embodiment, the other delivery area 24 is identical to
the delivery area shown in FIG. 3. The delivery area 24 includes a
circular base 40 that is positioned in an opening defined in a
plate 46 of the dock 22. The base 40 is sized to receive one of the
plates 36, and is constructed of a transparent material such as,
for example, glass, Plexiglas, or acrylic. The base 40 extends from
a top surface 42 to a bottom surface (not shown) positioned below
the plate 46. Because the base 40 is transparent, objects resting
on the top surface 42 of the base 40 are visible through the bottom
surface (i.e., from under the plate 46). A light-emitting diode
(LED) panel 50 is coupled to the bottom of the plate 46 and
configured to illuminate the objects resting on the top surface 42
of the base through the transparent base 40. In the illustrative
embodiment, the LED panel emits red light that is sufficiently
diffuse to minimize reflectance and has variable intensity that may
be controlled by an electronic controller 400 (see FIG. 14), as
described in greater detail below.
[0116] Each seed carrying plate 36 has a bin 44 defined therein
that receives the seeds 12. The dock 22 includes a plurality of
posts or guide pins 48 that surround the circular base 40. As shown
in FIG. 3, the pins 48 extend upwardly from the plate 46 and are
designed to support and/or secure the plate 36 on the base 40. In
other embodiments, the dock 22 may include other supporting
structure to guide, support, and/or secure the plate 36 on the base
40.
[0117] As indicated above, the system 10 is configured to locate
the seeds 12 on a plate 36 when the plate 36 is positioned on the
delivery area 24 or, more specifically, when the plate 36 is
positioned on the base 40. In the illustrative embodiment, a camera
52 is positioned above the delivery area 24, as shown in FIG. 1.
The camera 52 is electrically coupled to the electronic controller
400 (see FIG. 14) and is operable to capture images of the plate 36
and seeds 12. As described in greater detail below, the images are
sent to the controller 400 to determine the relative locations and
orientations of the seeds 12 on the plate 36 such that the system
10 can direct the robotic arm 16 to the seeds for processing. The
camera 52 may be embodied as any device suitable for capturing
images, such as a still camera, a video camera, or other device
capable of capturing video and/or images. Further, it will be
appreciated that an image captured by a camera may be described as
a projection of the scene in the field of view of the camera (e.g.,
the foreground objects and background) onto a plane perpendicular
to the optical axis of the camera.
[0118] Returning to FIG. 2, the dock 22 also includes a pair of
receiving areas 26 where seeds 12 may be positioned after
processing by the system 10. Like the delivery areas 24, each
receiving area 26 includes a plurality of posts or guide pins 48
that define an area sized to receive one of the plates 38. Each pin
48 extends upwardly from the plate 46, and the pins 48 cooperate to
support and/or secure the plate 38 in the receiving area 26.
[0119] As described above, the system 10 also includes an imaging
station 28 that is operable to capture a number of images of the
seeds 12, which used to determine the cutting planes for each seed
12. Referring now to FIGS. 4-5, the imaging station 28 includes a
lighted dome 54 and two cameras 56, 58 that are secured to the
table 20. The camera 56, 58 are electrically coupled to the
electronic controller 400 (see FIG. 14) and are operable to capture
images of the inner chamber 62 of the dome 54. In the illustrative
embodiment, the lighted dome 54 is an eight-inch diameter white LED
dome light manufactured by Advanced Illumination of Rochester, Vt.
The lighted dome 54 includes a concave interior wall 60 that
defines the bowl-shaped chamber 62 and a circular opening 64 that
permits access to the chamber 62.
[0120] As shown in FIG. 5, the dome 54 also includes a plurality of
LEDs 78 coupled to the wall 60 to light the chamber 62 during
operation. In the illustrative embodiment, the LEDs 78 are formed
as a ring of approximately 20 LEDs, which are sufficiently diffused
to prevent reflections onto objects within the dome 54 and may be
controlled by the controller 400 to vary the intensity of light
emitted from the LEDs 78. The ring is mounted around the upper
inside edge of the dome 54. It should be appreciated that in other
embodiments other lighting sources may be used.
[0121] The dome 54 includes a convex exterior wall 66 and a
plurality of legs 68 that extend downwardly from the wall 66 to the
table 20. The dome 54 has a lower opening 70 extending through the
walls 60, 66 at the apex of the convex exterior wall 66. In the
illustrative embodiment, a central axis 72 extends through the
centers of the upper opening 64 and the lower opening 70. Another
opening 74 extends through the walls 60, 66 on the side of the dome
54 facing the cameras 56, 58. The opening 74 has a longitudinal
axis 76 that extends orthogonal to the central axis 72.
[0122] Each of the cameras 56, 58 may be embodied as any device
suitable for capturing images, such as a still camera, a video
camera, or other device capable of capturing video and/or images.
The cameras 56, 58 include optical axes 80, 82, respectively, which
are aligned with the openings 70, 74 of the dome 54. In the
illustrative embodiment, the optical axes 80, 82 are parallel to
one another and perpendicular to the central axis 72 of the dome
54. As shown in FIGS. 4-5, the longitudinal axis 76 of the opening
74 is coincident with the axis 82 of the camera 58. Additionally,
in some embodiments, each of the cameras 56, 58 may include a lens
and be positioned such that, in a captured image of a seed 12
positioned within the lighted dome 54, the seed 12 is within at
least half of the field of view of the corresponding camera 56,
58.
[0123] The imaging station 28 includes an angled mirror 84 that is
positioned below the lower opening 70 of the dome 54. The angled
mirror 84 is configured to reflect light from the chamber 62 toward
the camera 56. In the illustrative embodiment, the surface 86 of
the mirror 84 is angled at a forty-five degree angle relative to
each of the central axis 72 and an optical axis 80 of the camera
56. As a result, light from the chamber 62 is reflected along the
optical axis 80 toward the camera 56. It should be appreciated that
in other embodiments the mirror may be omitted and the camera 56
positioned directly below the dome 54. Additionally, in other
embodiments, the camera 58 may be positioned adjacent to another
side of the dome 54. In still other embodiments, one of the cameras
56, 58 may be omitted.
[0124] In the illustrative embodiment, the imaging station 28
includes additional components to reduce the incidence of stray
light entering the dome 54 and improve the quality of imaging
performed at the imaging station 28. For example, a cover 90
positioned over the circular opening 64 of the dome 54 to reduce
the chance that stray light (e.g., from the environment of the
imaging station 28) enters the dome 54. As shown in FIG. 5, the
dome 54 includes a plurality of threaded bores 92 defined in the
rim 94 of the dome 54. Each bore 92 is sized to receive a
corresponding fastener 96 to secure the cover 90 to the dome
54.
[0125] The cover 90 includes a fabric sheet 100 that is secured to
a pad 102. The pad 102 is formed from a high-temperature flexible
silicon pad. In the illustrative embodiment, the pad 102 is black
such that it functions as a contrasting background to improve the
quality of images captured by the camera 56. It should be
appreciated that in other embodiments the pad may be made in
another contrasting color. In still other embodiments, the pad
and/or cover may be omitted from the imaging station 28. As shown
in FIG. 5, the cover 90 has a central opening 108 that permits the
robotic arm 16 to advance a seed 12 into the dome 54.
[0126] Another component to improve the quality of imaging is a
backstop 106 secured to the dome 54. As shown in FIG. 5, the
backstop 106 is positioned within the chamber 62 of the dome 54.
The backstop 106, like the pad 102, is configured to serve as a
contrasting background for images of the seed 12 captured by the
camera 58. It should be appreciated that in other embodiments the
backstop may be made in another contrasting color. In still other
embodiments, the backstop may be omitted from the imaging station
28. In yet other embodiments, the imaging station 28 may include an
environment for capturing images of the seeds 12 in addition or
alternatively to the lighted dome 54 such as, for example, another
lighted hollow-bodied structure, a planar monochromatic backdrop,
or some other suitable imaging environment.
[0127] As described above, the system 10 also includes a cutting
station 30 that is operable to cut each seed 12 based on the images
captured by the station 28. Referring now to FIG. 6, the cutting
station 30 includes a platform 110 and a cutting device 112
operable to cut the seed 12 on the platform 110. The platform 110
includes a pedestal 114 that extends upwardly from the table 20 and
a seed cutting block 116 secured to the upper end 118 of the
pedestal 114. The pedestal 114 is formed from a metallic material
such as, for example, stainless steel or aluminum. In the
illustrative embodiment, the cutting block 116 is formed from a
magnetic metallic material such as, for example, stainless steel.
It should be appreciated that in other embodiments the pedestal
and/or cutting block may be formed from other rigid materials such
as plastics, Teflon, or ceramics.
[0128] As shown in FIG. 7, the cutting block 116 is configured to
be removed from the pedestal 114 for sterilization or repair. In
the illustrative embodiment, the pedestal 114 includes a permanent
magnet 120 that is positioned adjacent to the upper end 118. When
the cutting block 116 is positioned on the pedestal 114, the magnet
120 exerts a force to retain the cutting block 116 on the pedestal
114. It should be appreciated that the magnet is not required to
retain the block 116 on the pedestal 114. In the illustrative
embodiment, the design of the pedestal 114 is sufficient to retain
the block 116 thereon.
[0129] In the illustrative embodiment, the cutting block 116 has a
body 122 and a flange 124 that extends outwardly from the body 122.
The lower end 126 of the body 122 has a substantially planar bottom
surface 128, and the body 122 has a substantially planar top
surface 130. A pair of angled surfaces 132, 134 extend upwardly
from the bottom surface 128. The angled surface 132 is connected to
a back surface 136, which extends vertically to the top surface
130. As shown in FIG. 7, the angled surface 132 and the back
surface 136 have a slot 138 defined therein.
[0130] As shown in FIG. 7, a groove 140 is defined in the upper end
118 of the pedestal 114, and the groove 140 is configured to
receive the lower end 126 of the block body 122. In the
illustrative embodiment, the groove 140 is defined by a
substantially planar surface 142 and a pair of angled surfaces 144,
146 that extend upwardly from the surface 142. In that way, the
configuration of the groove 140 substantially matches the
configuration of the lower end 126 of the block body 122.
[0131] The pedestal 114 also includes a rear wall 148 that faces
the back surface 136 of the cutting block 116 when the block 116 is
positioned in the groove 140. An alignment pin 150 extends
outwardly from the rear wall 148. The alignment pin 150 is sized to
be received in the slot 138 defined in the block 116 to ensure the
cutting block 116 is properly positioned on the pedestal 114.
[0132] As shown in FIGS. 7-8, the flange 124 of the cutting block
116 extends outwardly from the body 122 to a front wall 154. The
flange 124 includes a substantially planar upper wall 156 and a
substantially planar lower wall 158 that is positioned opposite the
upper wall 156. The upper wall 156 is sized to receive a soybean
seed 12. It should be appreciated that in other embodiments the
upper wall 156 may be resized according to the size of the seed to
be cut.
[0133] An opening 160 is defined in the front wall 154. A plurality
of inner walls 162 extend inwardly from the front wall 154 of the
flange 124 to define a slot 164 through each of the wall 156, 158.
As shown in FIG. 8, the slot 164 is centered in the flange 124, and
extends to a back edge 166 positioned between a rear edge 174 of
the flange 124 and the front wall 154. As described in greater
detail below, the slot 164 is sized to receive a cutting blade 170
when the blade is rotated vertically.
[0134] As shown in FIG. 9, the block body 122 has a substantially
planar side wall 172 that extends upwardly from the rear edge 174
of the flange 124 to an upper edge 176. In the illustrative
embodiment, the side wall 172 extends orthogonal to the upper wall
156. Another side wall 178 is connected to the upper edge 176 of
the side wall 172. The side wall 178 extends obliquely relative to
the walls 156, 172 to a top edge 180 connected to the top surface
130 of the block 116.
[0135] Returning to FIG. 6, the cutting station 30 also includes a
cutting device 112 that is operable to cut the seed 12 on the
platform 110. The cutting device 112 includes a support arm 190
configured to receive a cutting blade 170 and a drive assembly 192
configured to move the cutting blade 170 during the cutting
operation. The drive assembly 192 includes a drive stage 194 that
is secured to the table 20. The drive stage 194 includes a lower
body 196 and an upper body 198 configured to slide relative to the
lower body 196 in the direction indicated by arrows 200 in FIG. 6.
The drive stage 194 includes a linear drive electric motor (not
shown) that is electrically connected to the controller 400 and is
operable to move the upper body 198 relative to the lower body 196.
In the illustrative embodiment, the drive stage 194 is an Aerotech
model ANT95-50-L that has approximately 50 millimeters of
travel.
[0136] The drive assembly 192 of the cutting device 112 includes an
intermediate drive stage 210 that travels with the drive stage 194.
The intermediate drive stage 210 includes a base 212 that is
connected to the upper body 198 of the drive stage 194. The drive
stage 210 also includes a platform 214 that is moveably coupled to
the base 212. In the illustrative embodiment, the platform 214 is
configured to move vertically in the direction indicated by arrows
216 in FIG. 6. The drive stage 210 also includes a linear drive
electric motor (not shown) that is electrically connected to the
controller 400 and is operable to move the platform 214 relative to
the base 212. The drive stage 210 is illustratively embodied as
Aerotech model ANT95-3-V, which has approximately 3 millimeters of
travel.
[0137] As shown in FIG. 6, the drive assembly 192 includes a
rotational stage 220 that travels with the other stages 194, 210.
The rotational stage 220 includes a main body 222 that is connected
to the platform 214 of the drive stage 210. The rotational stage
220 also includes a mounting shaft 224 that is pivotally coupled to
the main body 222. An axis 226 is defined by the mounting shaft
224, and the shaft 224 is configured to rotate about the axis 226
in the directions indicated by arrows 228. In the illustrative
embodiment, the rotational stage 220 is connected to a source 230
of compressed air such as, for example, a compressor. The source
230 is electrically connected to the controller 400. When operated
by the controller 400, the source 230 may advance compressed air to
the stage 220 such that the shaft 224 is driven pneumatically about
the axis 226. The rotational stage 220 is illustratively embodied
as an EMI Plastics Equipment Swiveling Rotary, type RT25.
[0138] The support arm 190 of the cutting device 112 is secured to
the rotational stage 220. As shown in FIG. 6, the support arm 190
includes an elongated body 240 that has an end 242 secured to the
mounting shaft 224 of the stage 220. The support arm 190 also
includes a pair of jaws 244, 246 that are secured to the opposite
end 248 of the body 240. In the illustrative embodiment, each of
the jaws 244, 246 has an end 250 that is received in a channel 252
defined in the elongated body 240. The channel 252 defines a
longitudinal axis 254, and the jaws 244, 246 are configured to move
along the channel 252 toward and away from each other. In that way,
the jaws 244, 246 may be opened or closed. In the illustrative
embodiment, the support arm 190 is connected to a source 256 of
compressed air. The source 256 is electrically connected to the
controller 400. When operated by the controller 400, the source 256
may advance compressed air to the support arm 190 such that the
jaws 244, 246 are driven pneumatically along the channel 252. The
support arm 190 is illustratively embodied as an SMC MHZ2-20C1-M9PZ
gripper.
[0139] The jaws 244, 246 are configured to receive a cutting blade
170. Referring now to FIGS. 10-11, each cutting blade includes a
body 260 and a cutting edge 262 extending the length of the body
260. The cutting edge 262 is offset from the axis of rotation 226
when the cutting blade 170 is secured to the jaws 244, 246. The
body 260 also includes a pair of oblong mounting holes 264, which
are engaged by the jaws 244, 246 to secure the cutting blade 170 to
the device 112. The cutting blade 170 is illustratively formed from
a metallic material such as steel.
[0140] Each of the jaws 244, 246 extends from the end 250 to a tip
270. Each of the jaws 244, 246 includes an inner tab 272 positioned
along an inner edge 274 of the tip 270. Each tab 272 is sized to be
positioned in one of the holes 264 of a cutting blade 170. In the
illustrative embodiment, each of the jaws 244, 246 also includes a
slot 276 (see FIG. 10B) that is formed at the base of each tab 272.
In the illustrative embodiment, each slot is configured to capture
the blade and hold it level. As shown in FIGS. 11A and B, the blade
170 is advanced into the slots 276 when the jaws 244, 246 are moved
apart, thereby securing the blade to the jaws 244, 246. Each of the
jaws 244, 246 also includes an outer tab 278 that is positioned
along an outer edge 280 of the tip 270. The outer tab 278 includes
a beveled edge 282 to assist with alignment of the blade 170 as it
is inserted onto the jaws 244, 246.
[0141] As shown in FIG. 11A, the elongated body 240 of the support
arm 190 has a longitudinal axis 284. In the illustrative
embodiment, the cutting blade 170 is offset from the axis 284 when
secured to the jaws 244, 246. During operation, the offset of the
cutting blade 170 from the axis 284 lowers the cutting blade 170 to
reduce the risk that the cutting blade will contact the robotic arm
when cutting the soybean seed.
[0142] Referring now to FIG. 12, a tray 34 for holding unused
cutting blades 170 is positioned between the robotic arms 16. The
tray 34 includes a container 302 positioned above a light source
304. The container 302 is illustratively formed from a transparent
material such as, for example, Plexiglas. The container 302
includes a bottom wall 306 and a plurality of side walls 308 that
extend upwardly from the bottom wall 306. The walls 306, 308
cooperate to define a chamber 310 sized to receive unused cutting
blades 170.
[0143] In the illustrative embodiment, the light source 304 of the
tray 34 is positioned below the bottom wall 306. The light source
304 is operable to project light through the bottom wall 306 into
the chamber 310. The light source 304 is illustratively embodied as
a red light-emitting diode (LED). It should be appreciated that in
other embodiments other colored LEDs may be used. In still other
embodiments, other lighting sources may be used.
[0144] The system 10 includes a tray camera 312, which is mounted
above the tray 34. The camera 312 is operable to capture images of
the contents of the chamber 310. The camera 312 is electrically
coupled to an electronic controller 400 (see FIG. 14). As described
in greater detail below, the images may be sent to the controller
400 to determine the relative locations and orientations of the
blades 170 in the tray 34 such that the system 10 can direct the
robotic arm 16 to the blades 170 for retrieval.
[0145] Referring now to FIG. 13, each robotic arm 16 of the system
10 includes a grip assembly 320 configured to grasp and hold a
soybean seed 12. In the illustrative embodiment, the grip assembly
320 includes a body 322 that is attached to a distal section 324 of
each arm 16. The grip assembly 320 also includes a suspension
mechanism 326 that connects the body 322 to a grip 18. The body 322
has a proximal disk 328 that is secured to the distal arm section
324 and a plurality of posts 330 that extend from the disk 328 to a
distal disk 332.
[0146] The suspension mechanism 326 extends from a proximal end 334
that is secured to the disk 332 to a distal end 336. As shown in
FIG. 13, the grip 18 is secured to the distal end 336 of the
suspension mechanism 326. The suspension mechanism 326 is
configured to permit some axial movement of the grip 18, as
indicated by arrows 338, 340, such that the grip 18 may be advanced
into contact with a soybean seed 12 without crushing the seed. In
the illustrative embodiment, the suspension mechanism 326 includes
a biasing element such as, for example, a helical spring 342, that
biases the grip 18 outward, in the direction indicated by arrow
340.
[0147] The grip 18 of the assembly 320 is configured to grasp and
hold a seed 12. In the illustrative embodiment, the grip 18
includes a cylindrical body 350 that is secured to the distal end
336 of the suspension mechanism 326. The body 350 is formed from an
elastomeric material such as, for example, Viton, which is
commercially available from DuPont Corporation. It should be
appreciated that in other embodiments other elastomeric materials
may be used. The body 350 includes a bellows, which provides the
body 350 with limited flexibility. The body 350 also has a high
temperature rating to permit sterilization of the grip 18. In the
illustrative embodiment, the temperature rating is 446 degrees
Fahrenheit. It should be appreciated that in other embodiments
other elastomeric materials may be used.
[0148] The grip assembly 320 is configured to grasp and hold the
seed 12 via vacuum. To do so, the grip 18 includes a hollow
passageway 352 that extends longitudinally through the body 350
along an axis 358. The passageway 352 is connected to passageways
354 defined in the suspension mechanism 326 and the body 322 of the
grip assembly 320 and a negative pressure source 356. The negative
pressure source 356 is illustratively embodied as a pump and is
electrically coupled to the controller 400. The controller 400 may
operate the source 356 to draw a vacuum through the passageways
352, 354 and secure a seed 12 to the grip 18. In the illustrative
embodiment, the grip 18 has a radius of less than fifty percent of
the average length of a seed 12, which may vary depending on, for
example, the particular species of the seed 12.
[0149] As shown in FIG. 13, the grip assembly 320 also includes a
secondary cover 360 that is secured to the body 350. The secondary
cover 360 is designed to prevent stray light from entering the
lighted dome 54 during imaging of the seed 12. The cover 360
includes a bottom pad 362 formed from a black foam material and a
top pad 364 that is formed from black felt. In the illustrative
embodiment, the cover 360 is secured to the distal disk 332 via
adhesive. It should be appreciated that in other embodiments the
cover 360 may be secured with fasteners such as screws or bolts.
The cover 360 has a diameter of approximately 3.5 inches, which is
sufficient to enclose central opening 108 of the cover 90 of the
dome 54.
[0150] Referring now to FIG. 14, the system 10 includes an
electronic controller 400. The controller 400 is, in essence, the
master computer responsible for interpreting electrical signals
sent by sensors associated with the system 10 and for activating or
energizing electronically-controlled components associated with the
system 10. For example, the electronic controller 400 is configured
to control the operation of the cameras 52, 56, 58, 312, dome light
78, robotic arms 16, drive stages 194, 210, and so forth. While the
electronic controller 400 is shown as a single unit in FIG. 14, the
controller 400 may include a number of individual controllers for
the various components as well as a central computer that sends and
receives signals from the various individual controllers. The
electronic controller 400 also determines when various operations
of the system 10 should be performed. As will be described in more
detail below, the electronic controller 400 is operable to control
the components of the system 10 such that the system 10 selects and
processes soybean seeds 12 for use in transgenic protocols.
[0151] To do so, the electronic controller 400 includes a number of
electronic components commonly associated with electronic units
utilized in the control of electromechanical systems. For example,
the electronic controller 400 may include, amongst other components
customarily included in such devices, a processor such as a
microprocessor 402 and a memory device 404 such as a programmable
read-only memory device ("PROM") including erasable PROM's (EPROM's
or EEPROM's). The memory device 404 is provided to store, amongst
other things, instructions in the form of, for example, a software
routine (or routines) which, when executed by the microprocessor
402, allows the electronic controller 400 to control operation of
the system 10.
[0152] The electronic controller 400 also includes an analog
interface circuit 406. The analog interface circuit 406 converts
the output signals from the various components into signals that
are suitable for presentation to an input of the microprocessor
402. In particular, the analog interface circuit 406, by use of an
analog-to-digital (A/D) converter (not shown) or the like, converts
the analog signals generated by the sensors into digital signals
for use by the microprocessor 402. It should be appreciated that
the A/D converter may be embodied as a discrete device or number of
devices, or may be integrated into the microprocessor 402. It
should also be appreciated that if any one or more of the sensors
associated with the system 10 generate a digital output signal, the
analog interface circuit 406 may be bypassed.
[0153] Similarly, the analog interface circuit 406 converts signals
from the microprocessor 402 into output signals which are suitable
for presentation to the electrically-controlled components
associated with the system 10 (e.g., the robotic arms 16). In
particular, the analog interface circuit 406, by use of a
digital-to-analog (D/A) converter (not shown) or the like, converts
the digital signals generated by the microprocessor 402 into analog
signals for use by the electronically-controlled components
associated with the system 10. It should be appreciated that,
similar to the A/D converter described above, the D/A converter may
be embodied as a discrete device or number of devices, or may be
integrated into the microprocessor 402. It should also be
appreciated that if any one or more of the
electronically-controlled components associated with the system 10
operate on a digital input signal, the analog interface circuit 406
may be bypassed.
[0154] Thus, the electronic controller 400 may operate to control
the operation of the system 10. In particular, the electronic
controller 400 executes a routine including, amongst other things,
a control scheme in which the electronic controller 400 monitors
the outputs of the sensors associated with the system 10 and
controls the inputs to the electronically-controlled components of
the system 10. To do so, the electronic controller 400 performs
numerous calculations, either continuously or intermittently,
including looking up values in preprogrammed tables, in order to
execute algorithms to perform such functions as energizing the
robotic arms 16, activating the cameras 52, 56, 58, 312, energizing
the drive stages 194, 210, varying the light intensity of the LEDs
78 and LED panel 50 to improve image contrast, and so on.
[0155] In operation, the system 10 may be operated in accordance
with the exemplary procedure outlined in FIGS. 15-19 to
automatically select and process soybean seeds 12 for use in a
transgenic protocol. For example, the soybean may be prepared by
splitting the cotyledons of a seed 12 along the hilum to separate
the cotyledons. Removal of a portion of the embryonic axis leaves
part of the axis attached to the cotyledons prior to
transformation. The removal of the embryonic axis may be made by
trimming of the embryonic axis with the cutting device 112.
Typically, between 1/3 and 1/2 of the embryo axis is left attached
at the nodal end of the cotyledon
[0156] As shown in FIGS. 20-29, the system 10 engages in
preliminary steps to sterilize the grips 18 of the robotic arms 16,
select a cutting blade 170 for the cutting station 30, and capture
images of the seeds 12 located in the delivery areas 24.
Thereafter, the system 10 operates one of the robotic arms 16 to
pick-up a seed 12 from one of the delivery areas 24 and advance the
seed 12 to the imaging station 28, as shown in FIGS. 27-31. A
number of images may be captured by the imaging station 28, as
shown in FIGS. 32-55, before the seed 12 is advanced to the cutting
station 30. As shown in FIGS. 56-59, the cutting station 30 may be
operated to make one or more cuts to the seed 12 to prepare it for
transformation. The cut seed may then be advanced to one of the
receiving areas 26. A user may then remove the seed from the system
10 for further processing. The system 10 may then engage in a
number of cleaning and maintenance tasks before picking up and
processing another seed 12.
[0157] As shown in FIG. 60-62, a soybean seed 12 includes a pair of
cotyledons 412, 414, which are encased in a seed coat 416. The
soybean seed 12 has a longitudinal axis 418, which is defined along
its maximum dimension, and extends through opposite longitudinal
ends 420, 422 of the soybean seed 12. As shown in FIG. 60, the axis
418 extends between the cotyledons 412, 414.
[0158] The soybean seed 12 also includes a hilum 424 positioned
between the ends 420, 422 of the soybean seed 12. In the
illustrative embodiment, the hilum 424 includes an outer section
426 that is positioned outside of the seed coat 416 and an inner
section 428 that is positioned under the seed coat 416.
[0159] As shown in FIGS. 60-61, the hilum 424 is dorsally located
above the cotyledons 412, 414. The outer section 426 of the hilum
424 is positioned on a dorsal side 430 of the soybean seed 12. The
hilum 424 may also be viewed from the lateral side 432 (see FIG.
61) or the medial side 434 (see FIG. 5) of the soybean seed 12. As
shown in FIG. 61, the hilum 424 has a longitudinal axis 436 that
extends parallel to the overall longitudinal axis 418 of the seed
12. As shown in FIG. 60, the longitudinal axis 436 lies in a common
plane 438 with the axis 418 of the seed 12.
[0160] An embryonic axis 440 of the soybean seed 12 connects the
cotyledon 412 to the cotyledon 414. The embryonic axis 440 is
encased with the cotyledons 412, 414 in the seed coat 416. As shown
in FIG. 60, the embryonic axis 440, like the hilum 424, is centered
on the longitudinal axis 418 of the soybean seed 12. As shown in
FIG. 62, the embryonic axis 440 extends from a tip 442 positioned
above the inner section 428 of the hilum 424 to a base 444
positioned adjacent to the longitudinal end 420 of the seed 12. It
should be appreciated that in other embodiments the embryonic axis
440 may not overlap with the hilum 424 such that the axis tip 442
is spaced apart from the inner section 428 of the hilum.
[0161] Referring to FIG. 62, the internal structure of the soybean
seed 12 is shown in greater detail. The seed coat 416 includes a
thin outer layer 450 that surrounds the cotyledons 412, 414 and the
embryonic axis 440. The inner section 428 of the hilum 424 is
attached to the underside of the layer 450, while the outer section
426 of the hilum 424 is connected to an edge 452 of the outer layer
450. The embryonic axis 440 extends around a portion of the outer
circumference of the seed 12 from its tip 442 to its base 444
positioned adjacent to the seed end 420
[0162] Referring now to FIGS. 15-16, an illustrative operating
procedure 1000 for preparing the soybean seed 12 for transformation
with the system 10 is shown. It will be appreciated that prior to
commencement of the procedure 1000, the controller 400 may
calibrate the system 10, provide messages to the user, retrieve
user input, initialize safety mechanisms (e.g., a light curtain),
and perform other setup functions. For example, if not done
already, the controller 400 may calibrate the system 10 using any
suitable protocol to map or otherwise correlate the coordinate
system of the robotic arms 16 to the coordinate systems of the
various cameras 52, 56, 58, 312 such that locations of objects
captured in images may be translated to a location of that object
relative to the arms 16. Further, the controller 400 may provide
setup instructions to the user on a display 460 (e.g., to place the
plate 36 on the delivery areas 24), retrieve input from the user
via a user input device 462 (e.g., a desired trim depth of the
embryonic node of the seeds 12, a bisecting depth of the seeds 12,
etc.). The user input device 462 may be embodied as any integrated
or peripheral device such as a keyboard, mouse, touchscreen, and/or
other input devices configured to perform the functions described
herein.
[0163] In block 1002, the system 10 sterilizes the grips 18 of the
robotic arms 16. To do so, the controller 400 operates each robotic
arm 16 to insert its corresponding grip 18 into a container filled
with ethanol or another suitable sterilizing solution. The solution
illustratively contains 70% alcohol. The robotic arm 16 may be
operated to move the grip 18 up and down and side to side within
the ethanol for some period of time before advancing the grip 18
into a sterilizer 32, as shown in FIG. 20. In the illustrative
embodiment, each sterilizer 32 is a dry glass bead sterilizer such
as, for example, an InoTech BioScience Steri 250. The robotic arm
16 may again be operated to move the grip 18 up and down within the
sterilizer 32 for a few seconds in the illustrative embodiment. The
arm 16 may then withdraw the grip 18 from the sterilizer 32 such
that the grip 18 is permitted to cool.
[0164] Due to the heat generated by the sterilizer 32, the bellows
of the grip 18 may become stuck together such that performance of
the grip 18 may be impaired. To separate the bellows, the robotic
arm 16 may then move the grip 18 into contact with a flat sterile
surface, such as, for example, the top surface 130 of the cutting
block 116, as shown in FIG. 21. The controller 400 may then
activate the negative pressure source 356 to seal the grip 18 to
the cutting block 116. As shown in FIGS. 22-23, the grip 18 is
moved away from the cutting block 116 in 1 mm increments until the
suction is broken.
[0165] Returning to FIG. 15, the procedure 1000 may then advance to
block 1004. In block 1004, a cutting blade 170 is selected and
retrieved from the tray 34. To do so, the controller 400 operates
the camera 312 to capture images of the blades 170 in the tray 34.
One such image 500 is shown in FIG. 24. As shown in FIGS. 24-25,
the blades 170 may be positioned in arbitrary locations and
orientations relative to one another within the tray 34. The
controller 400 may process the captured image 500 to identify the
location 516 of one of the blades 170 in the tray 34, which may be
reflected by an analyzed image 518 as shown in FIG. 25.
[0166] For example, in the illustrative embodiment, the controller
400 utilizes a geometric object-identifying function of the
software package included with the Epson model C3 six-axis
articulated arms. In particular, a blade reference image (not
shown) loaded by the user and stored in the memory device 404 of
the controller 400 is compared to the captured image 500 of the
blades 170 to identify a match 502. The geometric
object-identifying function employs an algorithmic approach that
identifies matches to a reference image (i.e., an object model) by
using edge-based geometric features. Further, the geometric
object-identifying function includes various parameters such as a
reference image to be used for comparison to another image and an
acceptance or tolerance level required for the match 502. The
acceptance level corresponds with a likelihood of a match 502 and
may, without loss of generality, be considered herein as a
normalized value between 0 and 1. Accordingly, if the acceptance
level is set to 0.5, only those objects in the analyzed image
having at least a fifty percent likelihood of a match 502 with the
reference image based on a suitable imaging algorithm will be
identified by the controller 400. In a specific embodiment, the
acceptance level may correspond with a percentage of a reference
image that must be identified in a continuous region of an analyzed
image to constitute a match 502.
[0167] In the illustrative embodiment, the controller 400 analyzes
the captured image 500 using the matching algorithm, the blade
reference image, and a normalized acceptance level of 0.4 (i.e.,
400 out of 1000) to determine whether there are any blades 170 on
the tray 34. An assumption is made that if any blades 170 are on
the tray 34, even if the blades 170 are overlapping, such an
acceptance level should return the identified locations of those
blades 170 on the tray 34. As such, in another embodiment, a
different acceptance level may be used. If no blades 170 are
identified, the controller 400 determines that no blades 170 are
located on the tray 34 and processes the error. For example, the
controller 400 may instruct the user of the system 10 via a display
460 to place additional blades 170 on the tray 34 or otherwise
remedy the error.
[0168] If the controller 400 determines that at least one blade 170
is located on the tray 34, the controller 400 analyzes the captured
image 500 again with the acceptance level set to a higher threshold
value such as 0.95 (i.e., 950 out of 1000) to identify a blade 170
that does not overlap with another blade 170 on the tray 34. If at
least one non-overlapping blade 170 is identified, the controller
400 selects that blade 170 for use. However, if non-overlapping
blades 170 are identified, the controller 400 executes a protocol
to separate the overlapping blades 170.
[0169] In doing so, the controller 400 identifies the location of a
blade 170 that overlaps with another blade 170 on the tray 34. For
example, the controller 400 may use the image locations identified
with the normalized acceptance level set at 0.4, if saved, or
similarly analyze the image 500. When the group of blades 170 has
been identified, the controller 400 determines the geometric center
of the group using a suitable imaging algorithm (e.g., by detecting
a center of mass of the group) and instructs the corresponding
robotic arm 16 to move the grip assembly 320 into position for
grasping the group of blades 170 at the identified center of
mass.
[0170] To grasp an object from the tray 34 or plate 36, the grip
assembly 320 is positioned above a grip location or point 504 of
the object such that the hollow passageway 352 of the grip assembly
is approximately collinear with the point 504. The grip assembly
320 is then advanced downward toward the object until the grip 18
is in full contact with the outer surface of the object. As
described above, the suspension mechanism 326 operates to prevent
the object from being crushed while ensuring that the grip 18 is in
full contact with the object's surface to provide limited loss of
suction. The negative pressure source 356 may then be activated to
secure the object to the grip 18.
[0171] Similarly, if there are no non-overlapping blades, the grip
assembly 320 may grasp a group of blades 170 at the identified
center of mass. The controller 400 may then operate the arm 16 to
move the grip assembly 320 vertically a short distance (e.g., one
inch) above the surface of the tray 34 and horizontally a short
distance but still within a perimeter of the tray 34. The
controller 400 may then deactivate the negative pressure source 356
to drop the group of blades 170 back onto the tray 34. It will be
appreciated that one or more of the blades 170 within the group may
fall during transport. The controller 400 operates the camera 312
to capture another image of the blades 170 in the tray 34 and
analyzes the new image similarly to that described above to
identify a non-overlapping blade 170. If no non-overlapping blade
170 is identified, the controller 400 may again instruct the grip
assembly 320 to grasp a group of blades 170 and drop the blades 170
on another location within the tray 34. The controller 400 may
continue to repeat the routine until a non-overlapping blade 170 is
identified selected for use.
[0172] In another embodiment, the controller 400 may implement
other procedures for separating overlapping blades 170 and
identifying a particular blade 170 for selection. Further, the
controller 400 may utilize any suitable image processing algorithms
and techniques to identify the locations of the blades 170 in the
tray 34. For example, the controller 400 may utilize feature
detection algorithms, techniques, and filters such as Speeded Up
Robust Features (SURF), Scale-Invariant Feature Transform (SIFT),
Multi-Scale Oriented Patches (MOPS), Canny, image gradient
operators, and Sobel filters to identify features (e.g., interest
points such as corners, edges, blobs, etc.) of the image 500 and
the blade reference image. In some embodiments, the controller 400
may utilize feature matching algorithms such as the Random Sample
Consensus (RANSAC) algorithm to determine whether any features
identified in the image 500 and the blade reference image
correspond with one another and, if so, the corresponding locations
of those features. Additionally or alternatively, the controller
400 may utilize image segmentation algorithms (e.g., pyramid
segmentation, watershed algorithms, etc.) for identifying objects
in an image. It will be appreciated that, depending on the
particular embodiment, the controller 400 may utilize any one or
more of the algorithms described above during the analyses of
captured images.
[0173] After the controller 400 has identified a blade 170, the
controller 400 uses blade features such as, for example, the
mounting holes 264 of the blade 170 to locate the cutting edge 262
of the blade. The controller 400 may then calculate the rotation
angle of the blade 170 with respect to the grip assembly 320 and
calculate the correct position point 504 on the blade for
attachment of the grip 18. The grip assembly 320 grasps the blade
at the point 504 in a similar manner to that described above.
[0174] Returning to FIG. 15, the procedure 1000 advances to block
1006 once the grip 18 has picked up a blade 170. In block 1006, the
controller 400 operates the robotic arm 16 and the cutting device
112 to secure the cutting blade 170 to the cutting device 112. To
do so, the controller 400 activates the robotic arm 16 to move the
cutting blade 170 to the cutting station 30 and position the
cutting blade 170 above the jaws 244, 246 of the cutting device
112. To position the cutting blade 170 on the jaws 244, 246, the
robotic arm 16 may be moved in a circular motion to align the
oblong mounting holes 264 of the blade 170 with the tabs 272 of the
jaws 244, 246. The beveled edges 282 of the outer tabs 278 assist
in guiding the blade 170 onto the tabs 272. When the blade 170 is
positioned on the tabs 272, the grip 18 is moved downward, causing
the blade 170 to deflect slightly. As shown in FIG. 26, the
controller 400 may then operate the jaws 244, 246 to secure the
blade 170 to the cutting device 112. A camera (not shown) may be
used to capture images of the blade 170 positioned on the cutting
device 112, and the controller 400 may use image processing
techniques similar to those described above confirm the blade 170
is properly positioned on the jaws 244, 246.
[0175] With the blade 170 positioned on the jaws 244, 246, the
controller 400 may operate the compressed air source 256 to move
the jaws 244, 246 outward along the channel 252 of the elongated
body 240. As the jaws 244, 246 are advanced outwardly, portions of
the cutting blade 170 are advanced into the slots 276 formed at the
base of the tabs 272, thereby securing the cutting blade 170 to the
jaws 244, 246. The controller 400 may deactivate the vacuum source
356 to release the cutting blade 170 from the grip 18 and operate
the robotic arm 16 to move the grip 18 out of the cutting station
30.
[0176] Returning to FIG. 15, the procedure 1000 advances to block
1008 in which the controller 400 operates the camera 52 to capture
images of the seeds 12 on a plate 36 positioned in the
corresponding delivery area 24. One such image 510 is shown in FIG.
27. As shown in FIG. 27 and similar to that described above with
regard to the blades 170 in the tray 34, the seeds 12 may be
arbitrarily positioned relative to one another within the plate
36.
[0177] In block 1010, the controller 400 may process the captured
image 510 to determine the location of one of the seeds 12 on the
plate 36 for selection. To do so, the controller 400 may analyze
the captured image 510 using a matching algorithm (e.g., the
geometric object-identifying function described above) to compare a
reference image 512 of a seed 12 lying on its side, as shown in
FIG. 28, to the captured image 510. In the illustrative embodiment,
the controller 400 assumes that the user of the system 10 has
placed each of the seeds 12 on its side within the plate 36 in a
single layer. Accordingly, there is a high likelihood of detecting
a match 522. However, in other embodiments, the controller 400 may
not make such an assumption; rather, the controller 400 may, for
example, determine which seeds 12, if any, are not appropriately
oriented and ignore those seeds 12. The system 10 may generate a
warning or instruct the user to remedy the situation (e.g., via the
display 460), or otherwise handle the error. In other embodiments,
the controller 400 may use blob detection or other image analysis
algorithms to determine the location of the seeds 12 on the plate
36.
[0178] In any case, the controller 400 determines the locations 520
of one or more seeds 12 on the plate 36, which may be reflected by
an analyzed image 514 as shown in FIG. 29. Further, in some
embodiments, the controller 400 determines an angle of rotation of
the identified seed(s) 12 on the plate 36 relative to the seed 12
depicted in the reference image 512. Based on that information, the
controller 400 may determine an amount by which to rotate the grip
18 to place the secured seed 12 in a predefined orientation (e.g.,
zero degree angle relative to the coordinate system of the robotic
arm 16) on the grip 18. By doing so, the controller 400 may be able
to identify the hilum and embryo axis (i.e., the embryonic axis) of
the seed 12 as described below and save processing time.
[0179] Returning to FIG. 15, the procedure 1000 advances to block
1012. In block 1012, the controller 400 identifies and selects
(e.g., arbitrary or algorithmically) one of the seeds for trimming
and bisection by the system 10. In the illustrative embodiment, the
controller 400 identifies the center of mass of the selected seed
12 and uses that as the point 504 to attach the grip 18 as shown in
FIG. 30. In block 1014, the grip assembly 320 grasps the selected
seed 12 at its center of mass. To do so, the grip assembly 320 is
positioned above the center of mass (i.e., the point 504) such that
the hollow passageway 352 of the grip assembly is approximately
collinear with the point 504. The grip assembly 320 is then
advanced downward toward the selected seed until the grip 18 is in
full contact with the outer surface of the seed. As described
above, the suspension mechanism 326 operates to prevent the seed
from being crushed while ensuring that the grip 18 is in full
contact with the seed's surface to provide limited loss of suction.
The negative pressure source 356 may then be activated to secure
the seed to the grip 18. The procedure 1000 may then advance to
block 1016 in which the robotic arm 16 moves the gripped seed 12
through the central opening 108 in the cover 90 and into the
chamber 62 of the lighted dome 54, as shown in FIG. 31. In the
illustrative embodiment, the gripped seed 12 is positioned within
the chamber 62 at a location that is within the fields of view of
each of the cameras 56, 58 (e.g., an intersection point of the
optical axes 80, 82). For example, in some embodiments, the gripped
seed 12 is positioned, at least in part, within the focal plane of
each of the cameras 56, 58.
[0180] When the seed 12 is positioned in the chamber 62 of the
lighted dome 54, the procedure advances to block 1018, as shown in
FIG. 15. In block 1018, the controller 400 determines the proper
orientations of the gripped seed 12 for trimming and bisecting the
seed 12 with the cutting device 112. That is, the controller 400
determines how the seed 12 is positioned relative to the grip 18 so
that the robotic arm 16 can properly position the seed 12 on the
cutting block 116 for trimming and bisecting the embryo of the seed
12. To do so, an illustrative operating procedure 1200, as shown in
FIG. 17, may be used. Although the procedure 1200 is described
herein with regard to analyzing several still images in a linear
manner, it will be appreciated that, in some embodiments, the
controller 400 may perform multiple image analyses in parallel or
continuously analyze video, for example.
[0181] The procedure 1200 may begin with block 1202 in which the
controller 400 operates the camera 58 to capture an image 530 of
the gripped seed 12 from a side perspective. In block 1204, the
controller 400 analyzes the image 530 to determine whether a hilum
424 of the seed 12 is visible on the seed 12. That is, the
controller 400 determines whether the hilum 424 (see FIG. 32) is
within a field of view of the camera 58. To do so, the controller
400 may utilize any suitable image processing algorithms such as
those described herein. For example, in the illustrative
embodiment, the controller 400 utilizes a correlation model that
uses shadows (e.g., grayscale pixel intensity) to model the seed 12
and identify a match 534, if any, between the seed 12 and a
reference image 536 of a seed hilum as shown in FIG. 32. In
particular, the correlation model performs a pixel-to-pixel match
of the reference image 536 to the captured image 530.
[0182] In block 1206, the controller 400 determines whether the
hilum 424 of the seed 12 is within the field of view of the camera
58. If so, the procedure 1200 advances to block 1210. However, if
the controller 400 determines that the hilum 424 is not within the
field of view of the camera 58, the procedure advances to block
1208.
[0183] In block 1208, the controller 400 may reorient the seed 12
such that the hilum is within the field of view of the camera 58.
In particular, the controller 400 operates the robotic arm 16 to
rotate the seed 12 about the axis 358 of the grip 18 until the
hilum 424 is within the field of view of the camera 58. In some
embodiments, the robotic arm 16 rotates the seed 12 by an
incremental angle, the camera 58 captures a new image of the
gripped seed 12, and the controller 400 analyzes the new image to
determine whether the hilum 424 is now within the field of view of
the camera 58. If not, the routine may be repeated until the hilum
424 is within the field of view of the camera 58. In an embodiment,
the robotic arm 16 may first rotate the seed 12 by an angle of 180
degrees to expedite the process of locating the hilum 424. Once the
hilum 424 is determined to be within the field of view of the
camera 58, the procedure 1200 may advance to block 1210.
[0184] In block 1210, the controller 400 operates the camera 56 to
capture an image 540 of the gripped seed 12 from a bottom
perspective as shown in FIG. 33. The procedure 1200 then advances
to block 1212 of FIG. 18. In block 1212, the controller 400
analyzes the captured image 540 to identify a longitudinal axis 542
(i.e., a major axis) of the seed 12 in the image 540. In the
illustrative embodiment, the controller 400 utilizes a blob
detection algorithm to locate the seed 12 in the captured image 540
and determine the principal axes (i.e., the major axis and the
minor axes) of the seed 12. For example, the blob detection
algorithm may identify the seed 12 in the captured image 540 as a
blob, determine the center of mass and edges of that blob, and
approximate the major and minor axes based on that information.
[0185] It will be appreciated that the particular blob detection
algorithm utilized may vary depending on the particular embodiment.
For example, in the illustrative embodiment, the controller 400
uses blob detection algorithms of the software package included
with the Epson model C3 six-axis articulated arms. In some
embodiments, the blob detection algorithms may be based on
Difference of Gaussian (DoG), Laplacian of Gaussian (LoG), Hessian
determinants, and/or other operators. In an embodiment, the
controller 400 may utilize one or more of the blob detection
algorithms described in, for example, Lindeberg, Detecting Salient
Blob-Like Image Structures and Their Scales with a Scale-Space
Primal Sketch: A Method for Focus-of-Attention, 11(3) International
Journal of Computer Vision, 283-318 (1993). Further, in some
embodiments, the controller 400 may draw a rectangular border 548
around the seed 12 (or other objects) in a processed version of the
captured image 540 to indicate the location of the identified seed
12 (or other objects). In other embodiments, the controller 400 may
utilize other image analysis algorithms (e.g., image segmentation)
to identify the seed 12 and/or the longitudinal axis 542.
[0186] The controller 400 further determines an angle 544 of
rotation of the major or longitudinal axis 542 of the seed 12
relative to a horizontal axis 546 or other horizontal line 554 of
the captured image 540. In other words, the angle 544 defined
between the longitudinal axis 542 and the horizontal axis 546 or
other horizontal line 554 is determined. In the illustrative
embodiment, the camera 56 is configured to capture rectilinear
images; as such, the horizontal axis 546 of the captured image 540
may be considered parallel to an edge of the camera 56.
[0187] As shown in FIG. 34, the robotic arm 16 is capable of
reorienting the gripped seed 12 within the lighted dome 54. For
example, depending on the reorientation necessary, the robotic arm
16 may change the orientation of the seed 12 by rotating and/or
translating the seed 12. Accordingly, in block 1214 of FIG. 18, the
controller 400 operates the robotic arm 16 to orient the seed 12
such that the longitudinal axis 542 is parallel to the horizontal
axis 546 as shown in FIG. 35. In particular, the robotic arm 16
rotates the seed 12 about the axis 358. In some embodiments, the
controller 400 may not require precise parallelism but may
establish a tolerance for the angle 544. In some embodiments, the
tolerance may be less than or equal to 1.0 degree. In other
embodiments, the tolerance may be less than or equal to 0.5
degrees. In still other embodiments, the tolerance may be less than
or equal to 0.3 degrees for the angle 544. It should be appreciated
that similar tolerances may be established for any of the
measurements described herein. As indicated above, the camera 56
and the robotic arm 16 are calibrated such that their coordinate
systems are mapped to one another, so orienting the seed 12 in such
a way effectively aligns the longitudinal axis 542 of the seed 12
with an axis of the robotic arm's coordinate system.
[0188] Returning to FIG. 18, the procedure 1200 may advance to
block 1216 in which the controller 400 operates the camera 58 to
capture an image 550 of the gripped seed 12. As shown in FIG. 36,
the image 550 is a side elevation view of the seed from the
perspective of the camera 58. The image 550 may be analyzed in
block 1218 to identify the gripped seed 12 and a longitudinal axis
552 of the seed 12. It will be appreciated that the longitudinal
axes 552, 578 may or may not be coincident with one another due to
the irregular shape of the seed 12. The controller 400 may utilize
a blob detection algorithm to identify a location 556 of the seed
12 and/or locate the longitudinal axis 552 in the captured image
550 in a similar manner to that described above with regard to the
analysis of the captured image 540.
[0189] In the illustrative embodiment, the controller 400
identifies a left vertical slice 560 or cross section of the seed
12 at a left longitudinal end 562 of the seed 12 and a right
vertical slice 564 or cross section of the seed 12 at a right
longitudinal end 566 of the seed 12 in the captured image 550. As
shown in FIG. 37, each of the vertical slices 560, 564 is at least
one pixel in width. In the illustrative embodiment, the width of
the slices is 25 pixels, but the width may vary in other
embodiments. The controller 400 determines a center of mass 570 of
the left vertical slice 560 of the seed 12 and a center of mass 572
of the right vertical slice 564 of the seed 12. The longitudinal
axis 552 of the seed 12 in the captured image 550 is defined as the
line intersecting both centers of mass 570, 572. In other words,
the longitudinal axis 552 runs through the centers of mass of the
longitudinal ends 562, 566 of the seed 12. The controller 400
further determines an angle 574 of the longitudinal axis 552
relative to a horizontal axis 576 or other horizontal line 578 of
the captured image 550.
[0190] The procedure 1200 may advance to block 1220 in which the
seed 12 is reoriented. In particular, the controller 400 operates
the robotic arm 16 to orient the seed 12 such that the longitudinal
axis 552 is parallel to the horizontal axis 576 as shown in FIG.
38. Specifically, the robotic arm 16 rotates the seed 12 with
respect to the captured image 550 until the longitudinal axis 552
is parallel to the horizontal axis 576 (e.g., subject to a
tolerance level such as within one degree of parallelism). When the
seed 12 is properly oriented, the procedure 1200 continues to block
1222.
[0191] In block 1222, the controller 400 operates the camera 58 to
capture another image 580 of the gripped seed 12 from a side
elevation (i.e., the field of view of the camera 58), as shown in
FIG. 39. The image 580 is analyzed in block 1224 to identify the
gripped seed 12 and a location 596 of the hilum 424 of the seed 12
relative to a center of mass or longitudinal axis of the seed 12.
In the illustrative embodiment, the controller 400 utilizes blob
detection to determine the location 598 of the seed 12 in the
captured image 580. Additionally, the controller 400 utilizes a
suitable algorithm to determine the location of the hilum 424 on
the seed 12 in the capture image 580. For example, the controller
400 may determine the location 596 of the hilum 424 using the
reference image 536 of a hilum (see FIG. 32) and/or image feature
matching algorithms as described above. The controller 400
identifies a longitudinal end 582 (e.g., either the left or right
end) of the seed 12 and a vertical slice 584 of the longitudinal
end 582 of the seed 12 in a manner similar to that described above.
As shown in FIG. 40, the controller 400 identifies a center of mass
586 of the vertical slice 584 of the seed 12 and a center of mass
588 of the hilum 424 and draws a virtual line 590 between the
centers of mass 586, 588. The controller 400 further determines an
angle 592 of the line 590 relative to a horizontal axis 594 of the
captured image 580 or the longitudinal axis 552 of the seed 12.
[0192] Returning to FIG. 18, the procedure 1200 advances to block
1226 in which the controller 400 operates the robotic arm 16 to
orient the seed 12 to align the center of mass 588 of the hilum 424
with the longitudinal axis 552 of the seed 12 as shown in FIG. 41.
In particular, the robotic arm 16 rotates the seed 12 toward or
away from the camera 58 until the line 590 between the centers of
mass 586, 588 is parallel to the horizontal axis 594 of the
captured image 580. At that point, the line 590 corresponds to the
longitudinal axis 436 of the hilum 424 such that the seed plane 438
defined by the longitudinal axis 436 of the hilum 424 and the
longitudinal axis 436 of the seed 12 is aligned with a defined
plane of the coordinate system of the robotic arm 16.
[0193] The procedure 1200 may then advance to block 1228 of FIG.
19. In block 1228, the controller 400 operates the camera 58 to
capture images of the gripped seed 12 from a side elevation. Such
images 600 are shown in FIGS. 43-48. Returning to FIG. 19, in block
1230, the controller 400 analyzes the captured image 600 to
determine the location of the embryonic axis 440 of the seed 12.
The controller 400 may use any suitable algorithm for doing so.
[0194] For example, in the illustrative embodiment, the controller
400 may utilize a reference image 612 of an embryonic axis, as
shown in FIG. 42, in conjunction with the geometric
object-identifying function and/or correlation model described
above to identify the embryonic axis 440. It will be appreciated
that the controller 400 has identified a match 602 for the
embryonic axis 440 in each of FIGS. 46-48 as shown. However, the
controller 400 has failed to identify the embryonic axis 440 in
each of FIGS. 43-45, because a significant portion of the embryonic
axis 440 is not within the field of view of the camera 58. In those
circumstances, the controller 400 operates the robotic arm 16 to
rotate the seed 12 until the embryonic axis 440 is within the field
of view of the camera 58 and detected by the controller 400.
[0195] Returning to FIG. 19, the procedure 1200 advances to block
1232 in which the controller 400 determines a location at which to
trim the embryonic axis 440 of the seed 12. To do so, the
controller 400 determines the location 708 of the seed 12, the
location 602 of the embryonic axis 440, and the location 710 of the
hilum 424 of the seed 12 in the captured image 600 or a new image
captured by the camera 58 as shown in FIGS. 49-52. In particular,
the controller 400 identifies an edge 620 of the hilum 424 nearest
the embryonic axis 440 and an edge 622 of the seed 12 on the same
side as the embryonic axis 440 as shown in FIG. 51. Further, in the
illustrative embodiment, the controller 400 determines a vertical
cross section 624 halfway between the edges 620, 622. The vertical
cross section 624 corresponds with the location at which the system
10 is to trim the embryonic axis 440 of the seed 12. In other
embodiments, the controller 400 may identify a point other than the
midpoint between the edges 620, 622 (e.g., based on user
input).
[0196] As indicated above, the controller 400 has calibrated the
system 10 such that the coordinate system for the robotic arm 16
and the coordinate system of the camera 58 are mapped to one
another. Because the coordinate system of the robotic arm 16 is
known, the controller 400 knows the location of a center 626 of the
grip 18 with respect to the captured image 600. The controller 400
also knows the correspondence between physical distance in the
coordinate system of the robotic arm 16 (e.g., in millimeters) and
distance in the coordinate system of the camera 58 (e.g., in
pixels). That information is used to determine a horizontal
distance 628 between the center 626 and the vertical cross section
624 in the captured image 600 as shown in FIG. 52. The controller
400 further calculates a distance of the embryonic trimming cut
relative to the center 626 of the grip 18.
[0197] Returning to FIG. 19, in block 1234, the controller 400
determines the position at which to place the gripped seed 12 on
the cutting block 116 of the cutting station 30 to be trimmed and
bisected by the cutting blade 170. To do so, the controller 400
operates the camera 56 to capture an image 640 of the seed 12 from
a bottom perspective. As indicated above, the mapping between the
coordinate systems of the robotic arm 16 and the camera 56 is
known, so a point 642 projected along the grip axis 358 to the
captured image 640 may be determined. The controller 400 further
analyzes the captured image 640 to identify a back edge 644 of the
seed 12 (i.e., opposite the hilum 424 and the embryonic axis 440)
and a distance 646 between the point 642 and the back edge 644 as
shown in FIG. 53. Because the controller 400 has the location of
the front wall 154 of the cutting block 116 stored in memory, the
controller 400 is able to properly position the seed 12 on the
cutting block 116. In particular, the controller 400 operates the
robotic arm 16 to position the seed 12 on the flange 124 and with
the center of the grip 18 defined by the grip axis 358 positioned
the determined distance 646 away from the front wall 154.
[0198] Returning to FIG. 19, in block 1236, the controller 400
determines the depth of the trimming and/or bisection cut and the
positioning of the blade 170 for cutting the seed 12. As indicated
above, the controller 400 previously determined a point at which to
trim the embryonic axis (i.e., the vertical cross section 624 as
shown in FIG. 52). In the illustrative embodiment, the controller
400 maps the vertical cross section 624 to a corresponding location
650 on the captured image 640, which was taken from a different
perspective, by virtue of the known coordinate systems of each of
the cameras 56, 58. Further, the controller 400 determines a width
652 of the seed 12 at the corresponding location 650 as shown in
FIG. 54. The controller 400 also identifies the location of a back
edge 644 of the seed 12. Based on this information and the desired
depth of the trim cut and/or bisection cut (e.g., from user
inputs), the controller 400 is able to determine the distance to
move the cutting blade 170 toward the front wall 154 when trimming
and/or bisecting the embryonic axis 440.
[0199] The controller 400 also determines the appropriate
positioning of the cutting blade 170 for the bisection of the seed
12. To do so, the controller 400 operates the camera 58 to capture
an image 660 of the seed 12 and analyzes the captured image 660 to
locate a center of mass 662 of the embryonic axis 440 as shown in
FIG. 55. As indicated above, the controller 400 may first determine
the location 602 of the embryonic axis 440 in the captured image
660 using, for example, a feature matching algorithm in conjunction
with the reference image 612 (see FIG. 42). Further, the controller
400 determines a distance 664 between a bottom edge 666 of the seed
12 and the center of mass 662 of the embryonic axis 440. As
indicated above, the controller 400 may convert the pixel distance
to a physical distance. Accordingly, the distance 664 is used to
determine the distance above the flange 124 at which the horizontal
bisection cut is made.
[0200] Referring back to FIG. 15, once the controller 400
determines the proper orientations of the seed 12 for trimming and
bisection, the procedure 1000 advances to block 1020 of FIG. 16. In
block 1020, the controller 400 operates the robotic arm 16 to
position the gripped seed 12 on the cutting block 116. As described
above, based on structural data stored in memory, the controller
400 is able to determine the distance 646 between the grip axis 358
and the back edge 644 of the gripped seed 12. Accordingly, in the
illustrative embodiment, the controller 400 operates the robotic
arm 16 to position the seed 12 on the flange 124 at a point in
which the grip axis 358 is positioned the determined distance 646
away from the front wall 154 of the cutting block 116. At the
distance 646, the seed 12 is positioned for cutting at the proper
depth and orientation. In the illustrative embodiment, the seed 12
is positioned such that the back edge 644 of the seed 12 just
contacts the front wall 154 of the cutting block 116.
[0201] In block 1022, the controller 400 operates the cutting
device 112 to trim the embryonic axis 440. To do so, the controller
400 activates the compressed air source 230 to cause the shaft 224
(and hence the jaws 244, 246) to rotate about the axis 226. The
shaft 224 is rotated to position the cutting blade 170 vertically
(i.e., perpendicular to the flange 124 of the cutting block 116).
As shown in FIG. 56, the cutting blade 170 is aligned with the slot
164 defined in the flange 124.
[0202] The controller 400 may also operate the intermediate drive
stage 210 to raise or lower the cutting blade 170, as indicated by
arrows 700 in FIG. 56. To trim the embryonic axis 440, the
controller 400 operates the drive stage 194 of the cutting device
112 to advance the cutting blade 170 linearly along the axis 226
toward the seed 12 on the block 116. As shown in FIG. 57, the
cutting blade 170 is advanced into the slot 164 and the seed 12
until the cutting blade 170 reaches the previously determined
cutting distance (e.g., relative to the front wall 154), thereby
trimming the embryonic axis 440.
[0203] As shown in FIG. 63, the cutting blade 170 is advanced
through the embryonic axis 440 to separate the tip 442 of the axis
440 from the rest of the axis 440. As described above, typically,
between 1/3 and 1/2 of the embryonic axis 440 may left attached. In
other words, between 1/2 and 2/3 of the embryonic axis 440 may be
trimmed along with the tip 442 from the rest of the embryonic axis
440. In the illustrative embodiment, the cutting blade 170 does not
penetrate the cotyledons 412, 414 when the embryonic axis 440 is
trimmed. In some embodiments, it may be desirable to wound the
cotyledons 412, 414 by advancing the cutting blade 170 further into
the seed 10. The controller 400 may then operate the drive stage
194 to move the cutting blade 170 away from the seed 12 and out of
the slot 164.
[0204] The procedure 1000 may then advance to block 1024 in which
the controller 400 operates the cutting device 112 to position the
cutting blade 170 horizontally for bisection of the seed 12. To do
so, the controller 400 activates the compressed air source 230 to
cause the shaft 224 (and hence the jaws 244, 246) to rotate about
the axis 226 from the vertical position shown in FIGS. 56-57 to the
horizontal position shown in FIG. 58. The controller 400 may also
operate the intermediate drive stage 210 to raise or lower the
cutting blade 170 to align the cutting blade 170 with the
longitudinal axis 418 of the seed 12. As discussed above, the
controller 400 may use the distance 664 and other known physical
dimensions to determine the distance above the flange 124 at which
the cutting blade 170 is to be positioned.
[0205] In block 1026 of the procedure 1000, the controller 400
moves cutting blade 170 toward the front wall 154 to bisect the
seed 12. To do so, the controller 400 operates the drive stage 194
of the cutting device 112 to advance the cutting blade 170 linearly
along the axis 226 toward the seed 12 on the block 116. As shown in
FIG. 59, the cutting blade 170 is advanced into the seed 12 until
the cutting blade 170 reaches the previously determined bisection
distance as described above (e.g., relative to the front wall
154).
[0206] As shown in FIG. 64, the cutting blade 170 is aligned with
plane 438 defined by the longitudinal axis 436 of the hilum 424 and
the longitudinal axis 418 of the seed 12 and advanced through the
seed coat 416 and the hilum 424 along the plane 438, thereby
creating an opening 702 in the seed 12. The embryonic axis 440 is
sliced into a medial section 704 attached to the cotyledon 412 and
a lateral section 706 attached to the cotyledon 414. As shown in
FIG. 64, the cutting blade 170 passes through the base 444 of the
embryonic axis 440. It will be appreciated that, in the
illustrative embodiment, the cutting blade 170 does not completely
bisect the seed 12 into two pieces. Rather, after the embryonic
trimming and bisection, the seed 12 can still be transported by the
grip 18 as a single piece. The controller 400 may then operate the
drive stage 194 to move the cutting blade 170 away from the seed
12.
[0207] In block 1028 of the procedure 1000, the controller 400
operates the robotic arm 16 to move the bisected seed 12 to the
plate 38 located at the corresponding receiving area 26. The
controller 400 then deactivates the negative pressure source 356 to
drop the bisected seed 12 onto the plate 38. In block 1030, the
controller 400 operates the robotic arm 16 to clear any debris from
the cutting block 116. In some embodiments, the robotic arm 16 may
perform one or more passes of the grip 18 along the upper wall 156
of the flange 124 to clear debris. In other embodiments, the grip
assembly 320 includes a pressure source that is electrically
coupled to the controller 400 and configured to deliver pressurized
fluid (e.g., compressed air) through the passageways 352, 354 to
repel light objects such as debris. In such embodiments, the
controller 400 may deliver operate the pressure source to deliver
pressurized fluid to the cutting block 116 as the grip 18 passes
along the flange 124.
[0208] In block 1032, the controller 400 may operate the robotic
arm 16 and the cutting device 112 to replace periodically the
cutting blade 170. Depending on the particular embodiment, the
cutting blade 170 may be replaced after a predefined amount of time
has lapsed, after a threshold number of seeds 12 have been
processed, and/or in response to another condition.
[0209] It will be appreciated that the procedure 1000 or portions
of the procedure 1000 may be repeated for each seed 12 on the plate
36 in the delivery area 24. Further, the procedure 1000 may be
implemented using both robotic arms 16 such that the arms 16
alternate use of the stations 28, 30. Further, it should be
appreciated that the procedure may be implemented with one or more
robotic arms 16 that each utilize its own dedicated stations 28,
30.
[0210] After one or more of the cut seeds 12 have been placed in a
receiving area 26, the user may remove the seeds 12 from the system
10 for further processing. Among other things, the user may remove
separate the cotyledon from the seed coat, additionally wound the
cotyledon, or inoculate the cotyledon with an Agrobacterium
culture. To separate the seed coat 416 from the cotyledons 412,
414, the user may widen the opening 702 to further expose the
cotyledons 412, 414. The cotyledons 412, 414 may be removed from
the seed coat 416, and the seed coat 416 discarded. As shown in
FIG. 65, each cotyledon, which may be referred to as a split
soybean seed or cotyledon segment, includes a section of the
embryonic axis. In the illustrative embodiment, the cotyledon
segment 412 includes the section 704 of the embryonic axis 440,
while the cotyledon segment 414 includes the section 706 of the
embryonic axis 440. Each of the cotyledon segments 412, 414 is then
ready for further processing, including additional wounding or
inoculation with an Agrobacterium culture.
[0211] An Agrobacterium culture is a widely utilized method for
introducing an expression vector into plants is based on the
natural transformation system of Agrobacterium. Horsch et al.,
Science 227:1229 (1985). A. tumefaciens and A. rhizogenes are plant
pathogenic soil bacteria known to be useful to genetically
transform plant cells. The Ti and Ri plasmids of A. tumefaciens and
A. rhizogenes, respectively, carry genes responsible for genetic
transformation of the plant. Kado, C. I., Crit. Rev. Plant. Sci.
10:1 (1991). Descriptions of Agrobacterium vector systems and
methods for Agrobacterium-mediated gene transfer are also
available, for example, Gruber et al., supra, Miki et al., supra,
Moloney et al., Plant Cell Reports 8:238 (1989), and U.S. Pat. Nos.
4,940,838 and 5,464,763.
[0212] If Agrobacterium is used for the transformation, the DNA to
be inserted should be cloned into special plasmids, namely either
into an intermediate vector or into a binary vector. Intermediate
vectors cannot replicate themselves in Agrobacterium. The
intermediate vector can be transferred into Agrobacterium
tumefaciens by means of a helper plasmid (conjugation). The Japan
Tobacco Superbinary system is an example of such a system (reviewed
by Komari et al. (2006) In: Methods in Molecular Biology (K. Wang,
ed.) No. 343: Agrobacterium Protocols (2.sup.nd Edition, Vol. 1)
HUMANA PRESS Inc., Totowa, N.J., pp. 15-41; and Komori et al.
(2007) Plant Physiol. 145:1155-1160). Binary vectors can replicate
themselves both in E. coli and in Agrobacterium. They comprise a
selection marker gene and a linker or polylinker which are framed
by the right and left T-DNA border regions. They can be transformed
directly into Agrobacterium (Holsters, 1978). The Agrobacterium
used as host cell is to comprise a plasmid carrying a vir region.
The Ti or Ri plasmid also comprises the vir region necessary for
the transfer of the T-DNA. The vir region is necessary for the
transfer of the T-DNA into the plant cell. Additional T-DNA may be
contained.
[0213] The virulence functions of the Agrobacterium tumefaciens
host will direct the insertion of a T-strand containing the
construct and adjacent marker into the plant cell DNA when the cell
is infected by the bacteria using a binary T DNA vector (Bevan
(1984) Nuc. Acid Res. 12:8711-8721) or the co-cultivation procedure
(Horsch et al. (1985) Science 227:1229-1231). Generally, the
Agrobacterium transformation system is used to engineer
dicotyledonous plants (Bevan et al. (1982) Ann. Rev. Genet
16:357-384; Rogers et al. (1986) Methods Enzymol. 118:627-641). The
Agrobacterium transformation system may also be used to transform,
as well as transfer, DNA to monocotyledonous plants and plant
cells. See U.S. Pat. No. 5,591,616; Hernalsteen et al. (1984) EMBO
J 3:3039-3041; Hooykass-Van Slogteren et al. (1984) Nature
311:763-764; Grimsley et al. (1987) Nature 325:1677-179; Boulton et
al. (1989) Plant Mol. Biol. 12:31-40; and Gould et al. (1991) Plant
Physiol. 95:426-434.
[0214] Split soybean seeds comprising a portion of an embryonic
axis may be typically inoculated with Agrobacterium culture
containing a suitable genetic construct for about 0.5 to 3.0 hours,
more typically for about 0.5 hours, followed by a period of
co-cultivation on suitable medium for up to about 5 days. Explants
that putatively contain a copy of the transgene arise from the
culturing of the transformed split soybean seeds comprising a
portion of an embryonic axis. These explants may be identified and
isolated for further tissue propagation.
[0215] A number of alternative techniques can also be used for
inserting DNA into a host plant cell. Those techniques include, but
are not limited to, transformation with T-DNA delivered by
Agrobacterium tumefaciens or Agrobacterium rhizogenes as the
transformation agent. From example of Agrobacterium technology are
described in, for example, in U.S. Pat. No. 5,177,010, U.S. Pat.
No. 5,104,310, European Patent Application No. 0131624B1, European
Patent Application No. 120516, European Patent Application No.
159418B1 , European Patent Application No. 176112, U.S. Pat. No.
5,149,645, U.S. Pat. No. 5,469,976, U.S. Pat. No. 5,464,763, U.S.
Pat. No. 4,940,838, U.S. Pat. No. 4,693,976, European Patent
Application No. 116718, European Patent Application No. 290799,
European Patent Application No. 320500, European Patent Application
No. 604662, European Patent Application No. 627752, European Patent
Application No. 0267159, European Patent Application No. 0292435,
U.S. Pat. No. 5,231,019, U.S. Pat. No. 5,463,174, U.S. Pat. No.
4,762,785, U.S. Pat. No. 5,004,863, and U.S. Pat. No. 5,159,135.
The use of T-DNA-containing vectors for the transformation of plant
cells has been intensively researched and sufficiently described in
European Patent Application 120516; An et al, (1985, EMBO J.
4:277-284), Fraley et al, (1986, Crit. Rev. Plant Sci. 4: 1-46),
and Lee and Gelvin (2008, Plant Physiol. 146: 325- 332), and is
well established in the field.
[0216] Another known method of plant transformation is
microprojectile-mediated transformation wherein DNA is carried on
the surface of microprojectiles. In this method, the expression
vector is introduced into plant tissues with a biolistic device
that accelerates the microprojectiles to speeds sufficient to
penetrate plant cell walls and membranes. Sanford et al., Part.
Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299
(1988), Sanford, J. C., Physiol. Plant 79:206 (1990), Klein et al.,
Biotechnology 10:268 (1992).
[0217] Alternatively, gene transfer and transformation methods
include, but are not limited to, protoplast transformation through
calcium chloride precipitation, polyethylene glycol (PEG)- or
electroporation-mediated uptake of naked DNA (see Paszkowski et al.
(1984) EMBO J 3:2717-2722, Potrykus et al. (1985) Molec. Gen.
Genet. 199:169-177; Fromm et al. (1985) Proc. Nat. Acad. Sci. USA
82:5824-5828; and Shimamoto (1989) Nature 338:274-276) and
electroporation of plant tissues (D'Halluin et al. (1992) Plant
Cell 4:1495-1505).
[0218] While the disclosure has been illustrated and described in
detail in the drawings and foregoing description, such an
illustration and description is to be considered as exemplary and
not restrictive in character, it being understood that only
illustrative embodiments have been shown and described and that all
changes and modifications that come within the spirit of the
disclosure are desired to be protected.
[0219] There are a plurality of advantages of the present
disclosure arising from the various features of the method,
apparatus, and system described herein. It will be noted that
alternative embodiments of the method, apparatus, and system of the
present disclosure may not include all of the features described
yet still benefit from at least some of the advantages of such
features. Those of ordinary skill in the art may readily devise
their own implementations of the method, apparatus, and system that
incorporate one or more of the features of the present invention
and fall within the spirit and scope of the present disclosure as
defined by the appended claims.
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