Note: Descriptions are shown in the official language in which they were submitted.
APPARATUS AND METHOD FOR PREPARING
IMMATURE CORN EMBRYOS SUITABLE FOR
TISSUE CULTURE USING NON-JET FLUID FORCE
This application is a division of Canadian Serial No. 2,98 1,802 filed
June 1.2005.
FIELD OP TEE INVENTION
The present invention relates generallyto plant propagation and mechanical
methods for substantially isolating target plant tissues, each 06 emluris,
which are
suitable for genetic transfonnadon or tissue culture.
BACKGROUND OP THE INVENTION
The preptuation of tissues for plant propagation, regeneration and
ususibrmatIon is time consuming and labor intensive, capeoianY es it usually
involves
manna/ =felon of =storm)* or cub:arable plant tissues. For example, in corn
Pa max), individual immure embryo, are typically removed manually to provide
getierically-transformable expiate. The manual excision of emlnyogenic tissues
is
laborious and rinks ergonomic injury to the worker. Moreover, when larger
amounts
of tramfomnable plant tissue are required for high-throughput transformation
and
plant production, more workers most be employed and trained to meet the
increased '
demands. Additionally, there can be significant variability in the quality of
plant
tissues obtained, depending on the skill level, care, attentiveness, and
fatigue of the
individual workers. This tissue variability is emblematic, as poor quality
tissues
negatively impact the efficiency of subsequent tissue culture, genetic
liansfommtion,
and plant propagation. Thus, there is a need in the art for methods of
preparing target
plant tissues that are more rapid, reduce the overall ergonomic burden on
workers,
reduce the amount of workers needed to process the plant materials, and/or
yield plant
tissues that are of higher quality or more consistent quality than manually
produced
tissues.
CA 3 0 2 8 7 7 2 2 0 1 9 ¨1 0 ¨ 0 7
SUMMARY OF THE INVENTION
The present invention discloses methods and apparatuses to simplify, improve
safety,
increase reliability, reduce ergonomic injury, reduce the number of personnel
required, and/or
increase the speed with which target plant tissues are substantially isolated
for use in plant tissue
culture and genetic transformation. In particular, the present invention
discloses and claims
methods and apparatuses useful for substantially isolating embryos. In some
embodiments, the
methods and devices may be used to substantially isolate monocot embryos, such
as corn
embryos. The substantially isolated embryos are preferably suitable for
genetic transformation or
tissue culture. The methods and apparatuses disclosed herein are particularly
useful for high-
throughput processing (Le., substantially isolating large numbers of target
tissues and/or
processing large quantities of seeds).
One embodiment of the present invention provides an apparatus for preparing
immature
corn embryos suitable for tissue culture, comprising a means for contacting
immature corn
kernels having an opening in the pericarp of the kernel with a non-jet fluid
force, wherein the
fluid force contacts kernels and extracts embryos from the kernels, the
extracted embryos being
suitable for transformation or tissue culture.
One embodiment provides a method for preparing immature corn embryos suitable
for
tissue culture, comprising: applying a non-jet fluid force to immature corn
kernels having an
opening in the pericarp of the kernels.
The present invention also provides an apparatus for mechanically extracting
corn
embryos suitable for tissue culture from at least one immature corn ear
comprising: a handle
operable to hold the at least one immature corn ear; at least one solid
surface for applying
mechanical positive pressure to an exterior of at least one kernel on the at
least one immature
corn ear, wherein the mechanical positive pressure is sufficient to squeeze at
least one embryo
from the at least one kernel.
The present invention further provides an apparatus for mechanically
extracting corn
embryos suitable for tissue culture from at least one immature corn ear
comprising: (a) a portion
for rotating the at least one immature corn ear comprising (i) a handle
operable to hold a base of
the at least one immature corn ear, (ii) a connector operable to operatively
connect the handle to a
stepper motor; (iii) a stepper motor operable to rotate the at least one
immature corn ear such that
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CA 3028772 2019-10-07
at least one row of kernels contacts an extruder; (b) a portion for linearly
moving the
at least one immature corn ear; and (c) an extruder operable to apply force
sufficient
to squeeze at least one embryo and at least one endosperm from a pericarp. The
portion for rotating, the portion for linearly moving, and the extruder are
operatively
connected such that the apparatus is operable to extract at least one corn
embryo
suitable for tissue culture.
One aspect of this invention includes methods for substantially isolating
target
tissues from monocots, such as embryos, that are suitable for genetic
transformation
or tissue culture. The method comprises (a) providing monocot seeds containing
immature embryos that have an opening in the pericary' or seed coat of the
seeds; and
(b) applying force to the seeds sufficient to substantially isolate the
immature embryos
from the seeds. In sonic embodiments, immature corn embryos are substantially
isolated from corn seeds. The immature embryos thus obtained are preferably
suitable for genetic transformation or tissue culture.
Another aspect of this invention provides an apparatus for substantially
isolating target plant tissues, such as embryos, suitable for genetic
transformation or
tissue culture. The device comprises at least one aperture for guiding a fluid
stream.
In one embodiment, the fluid stream contacts kernels on an ear of coin and
causes the
embryos to become substantially isolated from the kernels. The substantially
isolated
embryos are preferably suitable for genetic transformation or tissue culture.
In one embodiment, the apparatus comprises at least one component selected
from among (a) at least one solid surface suitable for applying mechanical
positive
pressure to the exterior of a seed; (b) at least one aperture for guiding a
fluid flow; (c)
at least one aperture for applying negative fluid pressure; and any
combinations
thereof. The component may be used to direct a physical form on the seed
sufficient
to substantially isolate a target tissue, such as an embryo. Accordingly, the
apparatus
may be used to substantially isolate coal embryos suitable for genetic
transformation
or tissue culture from an ear of corn. In some embodiments, the aperture for
guiding a
2a
CA 3028772 2019-10-07
fluid flow directs the fluid flow to contact corn seeds or kernels on an ear
of corn. In
some embodiments, the aperture for applying negative fluid pressure directs
the
negative fluid pressure to contact corn seeds or kernels on an ear of corn.
The target
tissues substantially isolated by such an apparatus are preferably suitable
for tissue
culture or genetic transformation.
The invention further provides transgenic plants, plant tissues, and seeds.
Transgenic plant and plant tissues may be produced by (a) substantially
isolating a
target tissue using the methods and/or apparatuses described herein, (b)
introducing a
heterologous nucleic acid molecule into the target tissue to produce a
transformed
explant, and (c) culturing the transformed explant under suitable growth
conditions to
produce a transgenic plant tissue or plant. Any method of transformation is
suitable
and known to those of skill in the art. Additionally, suitable culture and
regeneration
conditions are known and routine. The transgenic plant, plant tissue, or seed
is
preferably a monocot, such as corn. The invention also includes all progeny
plants,
plant tissues, and seeds that are produced from the transgenic plant tissue or
plant.
Other embodiments of the invention are disclosed in the following detailed
description.
BRIEF DESCRH'TION OF THE DRAWINGS
Figure 1 depicts one embodiment of an apparatus of the present invention that
uses positive mechanical pressure for substantially isolating embryos, as
described in
Example 4.
Figure 2 depicts one embodiment of an apparatus of the present invention that
uses fluid jet positive pressure to dislodge embryos from seeds by a method of
the
invention, as described in Example 7. Legend: (A) robot with motion in X,Y and
Z
dimensions, (B) motor to rotate corn ear, (C) grasper, (D) handle to hold corn
ear, (E),
baffle to prevent material from splattering upwards, (F) flange to prevent
material
splattering upwards, (G) aperture for guiding fluid, (H) transparent tube, (I)
corn ear,
(j) shaking screen, (K) cheesecloth or other porous material, and (L) waste
container.
Figure 3 depicts one embodiment of a mounting mechanism using a magnetic
"handle" by which a corn ear can be secured to a robot arm, as described in
Example
7.
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CA 3028772 2019-01-02
Figure 4 depicts one embodiment of a nozzle useful in methods of the
invention, as described in Example 7. This nozzle generates a substantially
uniform,
flat sheet-like jet of fluid.
Figure 5 depicts one embodiment of an apparatus useful in methods of the
invention, as described in Example 13. This device includes a nozzle for
generating a
substantially flat fluid jet and an optional suction head. Figure 5 (top)
depicts a
cross-sectional view of one embodiment of such a device, showing how the
nozzle,
optional suction head, and corn ear may be positioned relative to each other.
Figure 5
(bottom) schematically depicts a corn ear positioned in the device. Legend:
(A)
base, (B) holder, (C) nozzle, (D) suction head, (E) corn ear, and (F) aperture
for
guiding fluid flow.
Figure 6 depicts one embodiment of a component useful for applying negative
fluid pressure useful in methods of the invention, as described in detail in
Example
13. Legend: (A) one or more apertures for guiding fluid flow.
Figure 7 depicts Figure 7A through 7C depict different views of an
embodiment of a device that uses a combination of forces and is useful in
methods of
the invention, as described in detail in Example 13. This device includes a
head with
a leading edge capable of applying a predefined amount of mechanical pressure
to the
base of kernels that previously have had the pericarp opened or truncated and
a
component for applying negative fluid pressure. This device can further
include a
means for dispensing fluid or for guiding fluid flow.
Figure 8 depicts an embodiment of the present invention in which a the top of
the cylinder is wholly, partially, or substantially covered with a membrane or
sheet of
soft material that is slightly smaller than the diameter of the corn ear; and
handle to
which it is attached.
Figure 9 depicts an alternative design to the embodiment depicted in Figure 8
in which small incisions are made in the membrane to provide additional
flexibility.
Figure 10 depicts a side view of the embodiments depicted in Figure 8 or 9 in
which a means is provided for securing the membrane on the appartus.
Figure 11 depicts an alternative membrane attachment system for use in the
present invention.
Figure 12 depicts a test of the supersoft 10A hardness silicone membrane
splash guard (1/32" thick) from McMaster-Carr # 9010K12. A 1" diameter hole
was
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CA 3028772 2019-01-02
made in the membrane. The cob was able to move both down through the opening
and also back up through it.
Figure 13 depicts a corn cob showing the location of the embryo within an
individual kernel. Also shown is one embodiment of the invention in which a
liquid
jet is directed to the basipetal side of the kernel, opposite the acropetal
side where the
embryo is located..
DETAILED DESCRIPTION OF THE INVENTION
Unless defined otherwise, all technical and scientific terms used herein have
= the same meaning as commonly understood by one of ordinary skill in the
art to
which this invention belongs, when taken in context of the present
specification.
Whom there are inconsistencies between the text of the specification and the
material
referred to herein, the definitions and meanings provided in the present
specification are intended. The nomenclature used herein and the manufacture
or
laboratory procedures described below are well known and commonly employed by
those of skill in the art.
The phrases "substantially isolated" or "extracted" refer to the processing of
a
target tissue (e.g., an embryo or other tissue explant) that resides in or
forms part of a '
larger tissue complex (e.g., a seed) such that the target tissue is physically
separated
from at least half of the larger complex. In some embodiments, a substantially
isolated target tissue may be physically separated from at least about 50%,
60%, 70%,
85%, 90%, 95%, 96%, 97%, 98%r 99% of the larger complex, or any fraction
thereof.
In other embodiments, the target tissue is physically separated from more than
about
80% to about 100%, about 90% to about 100%, or about 99% to about 100% of the
larger complex, or any fraction in between. In some embodiments, the target
tissue
may be physically separated from about 100% of the larger complex.
While a substantially isolated target tissue is physically separated from some
percentage of the larger complex, it does not necessarily have to be purified
from that
complex. In other words, the substantially isolated target tissue may remain
in a batch
with the larger tissue complex, so long as the target tissue is physically
separated from
the complex (as described above). In some embodiments, however, it may be
desirable to remove some or all of the separated complex from the
substantially
isolated target tissue. All such embodiments are within the scope of the
present
invention.
CA 3028772 2019-01-02
The phrase "target plant tissue" refers to a portion of a plant tissue or seed
that
one seeks to substantially isolate. In the present invention, target plant
tissue refers to
any portions of a plant or plant seed that can be substantially isolated and
used for
genetic transformation or tissue culture. In some embodiments, the target
plant tissue
is an embryo, in particular, an immature embryo from a monocot such as corn.
The phrase "suitable for genetic transformation" and "suitable for tissue
culture" refer to plant tissues that are competent for transformation or
growth in under
suitable plant culture conditions, respectively. One of skill in the art can
readily
determine if a particular target tissue is suitable for genetic transformation
or tissue
culture by using routine experimentation. For example, a sample from a batch
of
substantially isolated target tissues may be cultured on a suitable plant
media (also
known to those of skill in the art) to determine if the tissues are capable of
growth and
regeneration. Similarly, samples of substantially isolated target tissues can
be subject
to transformation and screened for the presence of a heterologous nucleic acid
molecule. Such techniques are routine and can rapidly identify which tissues
are
competent for transformation or tissue culture and which, if any, are not.
Where a term is provided in the singular, the inventors also contemplate
aspects of the invention described by the plural of that term.
Methods For Substantially Isolating Target Plgit Tissues
The present invention provides methods of substantially isolating target plant
tissues suitable for genetic transformation or tissue culture, comprising (a)
providing
seeds containing an opening in the pericarp or seed coat of the seeds; and (b)
applying
force to the seeds sufficient to substantially isolate a target plant tissue
from the seeds.
In some embodiments, the target plant tissue is an embryo. The embryos are
preferably monocot embryos, such as from corn. In some embodiments, the
substantially isolated target tissue may be isolated in whole are in part. For
example,
a batch of substantially isolated immature embryos may include intact embryos,
partial embryos, or mixtures thereof. Preferably, the intact and/or partial
tissues are
suitable for genetic transformation, tissue propagation, plant regeneration
and other
tissue culture applications.
As tissues are being isolated using, for example, streams of water, a
collection
receptacle may be provided. In some embodiments, it is useful to provide a
covering
for such collections vessels, in order to improve the efficiency of the
apparatus,
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CA 3028772 2019-01-02
reduce mess, prevent undesirable splashing from the jet stream, and/or limit
any
escape of extracted tissues during harvesting. Any suitable receptacle or
receptacle
covering may be employed. Examples are given elsewhere in this application and
are
also known to those of skill in the art.
Suitable coverings for the receptacle may include those made of metals, wood,
glass, meshes, fabrics, plastics, rubbers, latex, acrylics, and functionally
equivalent
materials. In some embodiments, the material is flexible so as to allow
penetration
and removal of an ear of corn while maintaining a substantially water tight
seal
around the ear during the extraction process. The material may be provided
with a
suitable opening to allow entry and removal of an ear of corn. In some
embodiments,
the material is solid and contains a flexible hole for receiving and holding
the ear
during extraction. In other embodiments, the material is flexible. Such
flexible
materials may be stretched over the receptacle to form a liquid tight fit, but
allows for
insertion of an ear either by penetrating the material or by providing an
opening to
receive the ear of corn. In still other embodiments, the material is a mesh or
screen
that has a flexible opening.
The coverings may be removable or semi-permanently attached. In some
embodiments, the materials are held by an elastic band or equivalent securing
means.
In other embodiments, the covering is held in place by weights, friction
collars,
hooks, snaps, or other functionally equivalent securing means.
The covering may be made of a flexible material and have varying thickness.
These factors may be varied in order to achieve the desired effects for
insertion and
extraction of ears of corn. The following table illustrates some hardness and
thickness
parameters for silicone coverings. The invention, however, is in no way
limited to
these few choices.
Durometer hardness Membrane thickness (in.) =
10A 1/32
10A 1/16
20A 1/32
20A 1/16
40A 1/32
40A 1/16
1.
In one embodiment, the collection receptacle is covered with a membrane or
sheet of soft material that is slightly smaller than the diameter of the corn
ear and
handle to which it is attached. In an alternative embodiment, small incisions
are
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CA 3028772 2019-01-02
made in the membrane to provide more flexibility. Figures 8-11 provide various
embodiments of the coverings and attachment means described above.
In some embodiments, the material is autoclavable. Autoclavable materials
are well known to those of skill in the art For example a soft material such
as a soft
silicone rubber sheet may be used. One of skill in the art is aware of the
possible
materials and physical arrangements that will permit extraction while
providing a
collection vessel that reduces escape of extracted tissue and prevents
undesirable
splashing.
Suitable procedures for plant tissue culture and regeneration are well known
in
the art. See, for example, United States Patent Number 5,550,318 to Adams
etal.,
United States Patent Number 5,780,708 to Lundquist at al., United States
Patent
Application Publication Number 2004/0210958 to Duncan etal., United States
Patent
Application Publication Number 2004/0016030 to Lowe etal., and United States
Patent Application Publication Number 2004/0244075 to Cai at at., which
disclose
transformation methods useful with corn, and United States Patent Application
Publication Number 2003/0024014 to Cheng etal., which disclose transformation
methods useful with wheat. These tissue culture
applications can include at least one process selected
from transformation, callus formation, direct embryogenesis, formation of
differentiated plant tissue, formation of at least one mature plant, formation
of at least
one fertile mature plant, and combinations of these processes. The plants
regenerated
from the extracted immature embryos may be regenerated, for example, through
differentiation of dedifferentiated tissue (calli) or by direct ernbryogenesis
of the
extracted immature embryos. Regenerated plants can preferably be grown to
maturity
to provide mature plants, and more preferably fertile mature plants. The
extracted
immature embryos and extracted non-embryo tissues may also be used for other
purposes, such as, but not limited to, genetic or biochemical analysis.
The methods and apparatuses of the present invention can be applied to any
monocot plants of interest. Preferred monocots include, but are not limited
to,
members of the family Poaceae, including grasses such as turf grasses and
grain crops
such as corn (maize), wheat, and rice. Particularly preferred monocots include
Zea
species, including corn (Zea mays), which has multiple kernels (seeds)
typically held
in rows on a corn ear.
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CA 3028772 2019-01-02
In general, the monocot seeds from which the target tissues are substantially
isolated are provided in any suitable manner. For example, seeds may be
attached to
the ear or head on which the seeds grow; in some embodiments the monocot seeds
may be removed from the ear or head prior to substantially purifying the
target tissue.
In some embodiments, an opening in the pericarp or seed coat of the monocot
seeds is Provided. This may be accomplished by any suitable technique, such
as, but
not limited to, making a hole, puncture, or incision with a needle, awl,
blade, or other
suitable implement. In some applications of the method, no pericarp tissue
need be
removed; in other embodiments, the opening of the pericarp may include removal
of
at least part of the pericarp and possibly of some non-embryo tissue (e.g.,
endosperm).
Preferably, the opening is sufficient to substantially separate the embryo
from the
seed. In some embodiments it may be necessary only to weaken the pericarp
sufficiently (for example, by abrasion, or by other physical, chemical, or
enzymatic
treatment) so that application of force to the seed results to substantial
isolation of the
target tissue, such as the embryo.
The method includes the step of applying force to the seeds sufficient to
substantially isolate the target tissue, such as an immature embryo, from the
seeds,
wherein the substantially isolated target tissue is suitable for genetic
transformation
and tissue culture. Force may be applied to multiple seeds consecutively or
simultaneously. The applied force can be continuous or non-continuous (for
example,
pulsed or wave-like force), and is generally mechanically applied, that is to
say, the
force is obtained through the use of a device or machine rather by human hand.
The
amount of force applied is preferably sufficient to overcome the adhesion of
the target
(e.g., embryo) and non-target (e.g., non-embryo tissue such as endosperm) from
each
other, thus allowing separation of the target and non-target tissues. Any
suitable force
or forces may be employed for removal of the target tissue from its seed, and
multiple
forces may be used in combination, sequentially or simultaneously. Suitable
forces
include, but are not limited to, fluid jet positive pressure, liquid jet
positive pressure,
mechanical positive pressure, negative pressure, centrifugal force, linear
acceleration,
linear deceleration, fluid shear, fluid turbulent flow, and fluid laminar
flow. Fluid
forces can be exerted by any fluid, gases or liquids or combinations of both.
Since a corn embryo is located on the acropetal side of a kernel it is
possible to
direct a liquid jet to the basipetal side of the kernel, if desired, to
successfully eject the
embryo (See, e.g., Fig. 13). In such an arrangement, the full force of the jet
is not
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CA 3028772 2019-01-02
directly impacting the embryo. Rather, the substantial amount of the force is
only
indirectly applied to the embryo itself. Thus, stronger forces may be applied
in the
apparatus to accelerate the removal of embryos without substantially
increasing the
damage to the embryos being removed.
Higher impact forces can be provided by forcing higher quantities of liquid
through the apparatus of the present invention. In some embodiments, however,
higher impact forces can be generated without using more liquid. For example,
in
some embodiments, the size of the jet opening is reduced so that the same
volume of
liquid can be used at a higher velocity. Since the energy of a moving object
is
proportional to the square of the velocity, a jet with the same volume can
have much
greater energy. A simple equation for kinetic energy of a moving object is
equal to
(1/2)(m)(v2). The calculation of the actual impact energy of a liquid jet
would also
take into account other factors known to those of skill in such art.
Additionally, some
embodiments may use a combination of increase fluid and changes in the size of
the
jet openings to achieve the desired force or energy.
Nozzles with gpm ratings of about 0.01 to about 0.25 may be used in the
present invention, or about 0.01 to about 0.2, or about 0.01 to about 0.1, or
about 0.01
to about 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.015, or any
whole number
or fraction in between these amounts. In some embodiments, using nozzles with
low
gpm ratings, like 0.033 or 0.021 gpm may be used. When such nozzles are used
with
higher pressure but directed at the opposite side of the kernel from the
embryo,
accelerated embryo harvesting may be achieved while avoiding injury to the
embryo.
In some embodiments, multiple jets are provided in the apparatus of the
present invention. Such an apparatus is useful to decrease the time needed to
harvest
the embryos from an ear of corn, hi some embodiments, the apparatus may have
2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16,17, 18, 19,20 or more jet openings
or
nozzles for conveying fluid force. In some embodiments, there are 2, 3,4, 5,
or 6 jet
openings or nozzles. In one embodiment, there are three openings. Such a
device is
depicted in Figure 12. In some embodiments, the openings are provided as
narrow-
angle flat stream jets, oriented horizontally. However, other embodiments of
the jet
openings are provided elsewhere throughout this application.
The method can further include-the step of separating the substantially
isolated
target tissue, such as immature embryos, from associated non-embryo tissue
such as
endosperm, glumes, and seed coat or pericarp tissues. Separation may be
CA 3028772 2019-01-02
accomplished by one or more suitable techniques, including, but not limited
to,
separation by size exclusion (for example, by filtration in one or more
filtering steps),
separation based on hydrophobicity, hydrophilicity, lipophilicity, or other
attractive
forces, and separation by mass or density differentials (for example,
separation by
centrifugation, settling, and decanting). The separation step or steps can be
optional,
for example, where no additional isolation of intact or partial embryos is
necessary for
their use in tissue culture.
The method of the invention is particularly suitable to applications where a
large number of target tissues must be provided, for example, in high-
throughput
processes or screening, or in batch processing for genetic transformation or
tissue
culture. Automation of the method is possible, for example, by employing
robotic or
mechanical handling of the corn ears or seeds, opening of the pericarp,
application of
force to the seed, or the optional separation steps. Such automation may use
optical
or mechanical sensors to aid in positioning the corn ears or seeds relative to
the
applied force or forces, or in the separation steps. In one preferred
embodiment, the
method provides substantially isolated embryos at a rate of between about 250
to
100,000 or more embryos per employee-day; or between about 250 to about
100,000,
or about 250 to about 50,000, or about 250 to about 20,000, or about 250 to
about
10,000, or about 250 to about 5000, or about 250 to about 3000, or about 250
to about
1000 embryos per employee-day; or between about 800 to about 100,000, or about
800 to about 50,000, or about 800 to about20,000, or about 800 to about
10,000, or
about 800 to about 5000, or about 800 to about 3000, or about 800 to about
1000 =
embryos per employee-day; or between about 2500 and about 100,000, or about
2500
to about 50,000, or about 2500 to about 20,000, or about 2500 to about 10,000,
or
about 2500 to about 5000, or about 2500 to about 3000 embryos per employee-
day; or
between about 5000 and about 100,000, or about 5000 to about 50,000, or about
5000
to about 20,000, or about 5000 to about 10,000 embryos per employee-day, or
any
fraction or whole number in between any of the aforementioned ranges. As a
reference, an employee-day is equivalent to one day's labor of one employee of
average skill in the art. While average employee output can vary; the
following is
given as a guideline for purposes of comparing the present invention with the
current
average employee output. To manage the ergonomic burden, it is currently
suggested
that workers excise about one ear of corn per a day (approximately 200 to 300
embryos) about twice per week. Thus, an average employee following such
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CA 3028772 2019-01-02
recommendations can produce up to about 600 embryos per week. It is possible
that
an average employee could produce up to about 500-800 excised embryos in one
day.
However, maintaining such an output over the course of several days or even
weeks is
not recommended due to the increased ergonomic burdens and quality concerns.
As
noted above, the present invention overcomes these significant output
limitations.
Apparatuses for Substantially Isolating Target Plant Tissues
The present invention also provides apparatuses for substantially isolating
target tissues, such as corn embryos, that are suitable for genetic
transformation or
tissue culture. In an embodiment for separating corn embryos, such an
apparatus
comprises at least one aperture for guiding a fluid stream, wherein the fluid
stream
contacts kernels on the corn ear and substantially isolates embryos from the
kernels.
Generally, it is preferred that the fluid stream contact as many of the
kernels in a
given period of time as is convenient, so as to more rapidly isolate embryos.
The at
least one aperture can include a single aperture or multiple apertures (for
example,
single or multiple nozzles, which can include flat, round, oval, fan-shaped or
other
patterned nozzles, and adjustable, moving, or stationary nozzles), and can
generate a
fluid flow of any suitable type and medium. Fluids may be gases (such as air,
nitrogen, or gas mixtures), liquids (such as water, physiological saline, or
various
culture media), or combinations. Suitable fluid flows include, but are not
limited to,
fluid jets (such as single or multiple columnar jets; flat, cone-shaped, or
fan-shaped
jets or sprays; and sheet-like jets), laminar fluid flow, and turbulent fluid
flow.
Suitable fluid flows can result in a variety of forces to remove the embryo
from its
kernel, including positive pressure or negative pressure or both; such forces
can be
uniform or non-uniform, continuous or non-continuous (such as a pulsed or wave-
like
force), or in any combination thereof.
The apparatus of the invention may further include a means for moving the
target tissue being substantially purified and the fluid stream, relative to
each other.
For example, either the ear of corn containing seeds or the fluid stream, or
both, may
be moved. Various embodiments of the apparatus can be used with single or
multiple,
intact or partial ears of corn. For example, the corn ear or ears can be
secured to a
holder or grasper, which is moved relative to the fluid stream. In other
embodiments,
however, the corn ear or ears need not be individually secured to a holder but
can be
freely movable so as to allow multiple kernels to be contacted by the force
used to
12
CA 3028772 2019-01-02
remove the embryos from the kernels. The means for moving at least one corn
ear
relative to the fluid stream can rotate the at least one corn ear and the at
least one
aperture relative to each other, or can move the fluid stream along the
longitudinal
axis of the at least one corn ear, or can provide any suitable three-
dimensional
movement of the at least one corn ear and the at least one aperture relative
to each
other, such as a combination of rotation and longitudinal motion.
The apparatus of the invention can further include at least one separator for
separating target tissues from non-target tissues. For example, embryos may be
separated from non-embryo tissues, wherein the separated embryos comprise at
least
some corn embryos suitable for genetic transformation or tissue culture.
Separators
can work by any suitable mechanism, including, but not limited to, separation
by size
exclusion (for example, using a mesh, screen, perforated surface, or other
device
capable of excluding objects of a certain size), separation based on
hydrophobicity or
other attractive forces (for example, using a material, solid or fluid, that
can attract or
repel the embryos), and separation by mass or density differentials (for
example,
using a centrifuge, or using solutions for differential settling). In certain
embodiments, the at least one separator can be optional, for example, where no
additional isolation of intact or partial embryos is necessary for their use
in genetic
transformation or tissue culture.
The substantially isolated (and optionally separated) immature embryos
include at least some embryos, such as immature intact or partial embryos,
suitable
for tissue culture applications, transformation, callus formation, direct
embryogenesis,
formation of differentiated plant tissue, formation of at least one mature
plant,
formation of at least one fertile mature plant, and combinations of these
processes, as
described above. The substantially isolated immature embryos and non-embryo
tissues may also be used for other purposes, such as, but not limited to,
genetic or
biochemical analysis.
The present invention further provides an apparatus for mechanically
substantially isolating multiple cam embryos suitable for genetic
transformation or
tissue culture from at least one immature corn ear, including at least one
component
selected from (a) at least one solid surface for applying mechanical positive
pressure
to the exterior of kernels on the at least one immature corn ear; (b) at least
one
aperture for guiding a fluid flow, wherein the fluid flow contacts kernels on
the at
13
CA 3028772 2019-01-02
least one immature corn ear; and (c) at least one aperture for applying
negative fluid
pressure, wherein the negative fluid pressure contacts kernels on the at least
one
immature corn ear; and wherein the at least one component applies force to the
kernels sufficient to substantially isolate embryos from the kernels, the
substantially
isolated embryos including multiple immature embryos suitable for genetic
transformation or tissue culture. A suitable apparatus applies one or more
forces
sufficient to substantially isolate the immature embryos from the seeds,
wherein the
substantially isolated immature embryos include embryos suitable for genetic
transformation or tissue culture. The one or more forces may be applied to
multiple
seeds consecutively or simultaneously, in a continuous or non-continuous
manner,
and is generally applied mechanically and not manually. Multiple forces may be
used
in combination, sequentially, or simultaneously. Suitable forces include, but
are not
limited to, fluid jet positive pressure, liquid jet positive pressure,
mechanical positive
pressure, negative pressure, centrifugal force, linear acceleration, linear
deceleration,
fluid shear, fluid turbulent flow, and fluid laminar flow. Fluid forces can be
exerted
by any fluid, gases or liquids or combinations of both.
Combination apparatuses of the invention can optionally include a means for
moving the at least one corn ear relative to the source or sources of force
(that is to
say, the solid surface for applying mechanical positive pressure, the aperture
for
guiding a fluid floiv, or the aperture for applying negative fluid pressure).
Preferably
the ear or ears is moved relative to the source of force so that the force or
forces
contact as many of the kernels in a given period of time as is convenient, so
as to
more rapidly isolate embryos.
Combination apparatuses of the invention can further include at least one
means for further separation of the substantially isolated immature embryos
suitable
for genetic transformation or tissue culture, wherein the separated embryos
comprise
at least some corn embryos suitable for genetic transformation or tissue
culture.
Separators can work by any suitable mechanism, including, but not limited to,
separation by size exclusion, separation based on attractive forces, and
separation by
=
mass or density differentials.
Transformed Plants and Methods of Their Production
The present invention also provides a transformed monocot plant, produced by
the steps including (a) providing at least one transformable target tissue
using the
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CA 3028772 2019-01-02
methods or apparatuses described herein; (b) introducing a heterologous
nucleic acid
molecule into the transformable target tissue to produce a transformed
explant; and (c)
growing a transformed monocot plant from the transformed explant. Preferred
monocots of the invention are transformed members of the family Poaceae,
including
grasses such as turf grasses and grain crops such as corn (maize), wheat, and
rice.
Particularly preferred monocot plants include transformed Zea species, such as
Zea mays. Transformed corn preferably contains at least one heterologous
nucleic
acid molecule capable of conferring a desired trait to the transformed corn,
such as
herbicide resistance, pest resistance, cold germination tolerance, water
deficit
tolerance, increased productivity, increased yield, and the like. Practical
transformation methods and materials for making transgenic monocot plants of
this
invention (for example, various media and recipient target cells,
transformation of
immature embryos, and subsequent regeneration of fertile transgenic plants)
are
disclosed, for example, in United States Patent Number 6,194,636 to McElroy et
al.,
United States Patent Number 6,232,526 to McElroy et al., United States Patent
Application Publication Number 2004/0216189 to Houmard at al., United States
Patent Application Publication Number 2004/0244075 to Cai et al., which
disclose
methods useful with corn, and United States Patent Application Publication
Number
2003/0024014 to Cheng et al., which discloses methods useful with 'wheat.
Single or multiple heterologous nucleic
acid molecules may be used for transforming the monocot plants of the
invention; for
example, constructs for coordinated decrease and increase of gene expression
are
disclosed in United States Patent Application Publication Number 2004/0126845
to
= Van Eenenna.am et al.
The seedi of resulting transgenic, fertile plants of the invention can be
harvested and used to grow progeny generations, including hybrid generations,
of
transformed plants that include the heterologous nucleic acid molecule in
their
genome. Thus, the present invention includes both primary transformed plants
("RO"
plants, produced by transforming embryos provided by a method of invention)
and
their progeny carrying the heterologous nucleic acid molecule. Such progeny
transgenic plants can be prepared by crossing a transformed monocot plant of
the
invention having the heterologous nucleic acid molecule with a second plant
lacking
the construct. Also, a transformed monocot plant of the invention can be
crossed with
a plant line having other heterologous nucleic acid molecules that confers
another trait
CA 3028772 2019-01-02
to produce progeny plants having heterologous nucleic acid molecules that
confer
multiple traits.
16
CA 3028772 2019-01-02
EXAMPLES
Example 1: Method to Extrude Multiple Corn Embryos
This example shows a method using mechanical positive pressure from an
extruder device to produce embryos suitable for tissue culture or genetic
transformation.
The tops of kernels were sterily removed from an immature ear of corn (Zea
mays) with a common vegetable peeler. The peeler was pushed from the basal end
of
the corn ear to the apical end using a slight sawing motion to obtain a quick,
sharp
truncation of the kernels. While in this embodiment the individual kernels are
truncated to expose the interior tissues, in other embodiments, it may be
necessary
only to ensure that an opening (such as a puncture or incision or abrasion) is
made in
the pericarp without actual removal of pericarp material. Where intact embryos
are
desired (for example, intact embryos for transformation), the size of any
opening is
preferably sufficient to allow removal of the embryo without damaging it.
Opening of
the pericarp can be accomplished by using any suitable device, including, but
not
limited to, blades and abrasive materials. For example, a vegetable peeler is
designed
to be relatively safe and fast to use; it has a regulated cutting depth and
also requires
less skill to use than a scalpel. Other tools with similar functions can be
employed.
The devices for opening the pericarp are preferably sterilizable, for example,
by
autoclaving or heating or by chemical sterilization. These pericarp treatment
processes can be automated; for example, a blade or blades or abrader can be
motorized.
A sterile extruder (in this case, a 4 millimeter diameter rod) was pushed
against the base of the truncated kernels. Other suitable extruding devices
may be
employed. Preferably, such devices should have a size and shape capable of
applying
a relatively localized force to the base of the truncated kernels to eject the
embryos
and endosperms. Preferably, the force applied is of sufficient magnitude and
is
applied in a suitable direction such that the advancing extruder does not
"ride up"
over the forward kernels. The trailing edge of the extruder preferably also
provides a
surface on which the ejected embryos and endosperms accumulate; for example, a
flat
piece of stainless steel with a rounded front edge could be used. In this
example, the
embryos were gently squeezed out from the pericarp, followed by the
endosperms.
17
CA 3028772 2019-01-02
The extruded embryos and endosperms came to rest on the top of the advancing
extruder rod, and were not crushed during the process.
The mixture of embryos and endosperms was washed with an aqueous fluid
medium (water, liquid medium, or saline) onto a sterile mesh having diamond-
shaped
openings (about 2 x 3 millimeters). The endosperms were observed to be largely
retained, and the smaller embryos and some smaller endosperm debris were
washed
through the screen into a collecting receptacle. The collected embryos were
washed
twice to remove small debris.
The washed embryos were further purified by a flotation process. In the first
step of the flotation process, the aqueous fluid medium was thoroughly
withdrawn
from the collecting receptacle, which was allowed to dry briefly (for example,
about a
minute), such that remaining aqueous medium withdrew from the waxy surface of
the
embryos, exposing them directly to the air. New aqueous medium was added, and
the
majority of the embryos floated because their waxy surface was not rewetted by
the
fluid. Non-embryo tissues such as endosperm debris remained submerged in the
medium, and a clear separation of embryos and non-embryo tissues was obtained.
The flotation of the extruded embryos could be improved by more rapid,
complete, or
reproducible withdrawal of the aqueous medium, such as through the use of
aspiration, or by capillary action (e. g., of a sterile absorbent placed in
the collecting
receptacle to absorb the fluid away from the extruded embryos).
A yield of approximately 100 embryos was isolated in this preliminary
experiment, wherein only a portion of the embryo-endosperm material from the
entire
ear was processed. These results demonstrate that methods of the present
invention
are practical and convenient for harvesting large numbers of immature embryos
from
corn cobs.
The embryos isolated by a method of the invention may then be used in tissue
culture procedures, for example, regeneration methods to generate transgenic
corn
= plants. Transfer of the isolated embryos to culture medium was easily
done by
placing forceps, with the tips closed together, underneath the floating
embryos, lifting
them free of the liquid with the forceps and placing them on culture medium.
Another
technique could be to pick the isolated embryos up with an instrument that has
a
hydrophobic surface. An additional technique would be to transfer embryos by
hydrophobicity, for example, transferring them to the medium surface by a
small puff
18
CA 3028772 2019-01-02
of air or sudden mechanical movement, such that their kinetic energy exceeds
the
hydrophobic force that holds them to the instrument.
Example 2: Visual Confirmation of Embryo Size
This example describes an improvement to one embodiment of the method of
the present invention, as described in Example 1. Using the approach described
in
Example 1, inunature corn embryos need to be as close to the truncated part of
the
kernel in order to be ejected in the greatest numbers. Variation in immature
corn
embryo size is an important consideration in gauging the amount of kernel top
to
remove. Embryos tend to be largest in the mid-section of the ear, with
somewhat
smaller embryos towards the ends. Smaller embryos, e. g., smaller than about
1.5
millimeters in length are more difficult to remove unless they are close to
the
truncation.
One way to ensure that enough of the kernel has been decapitated above
embryos of varying sizes is to observe the cob during the decapitation process
under
low magnification. For example, low magnification goggles (Donegan Opti-
VISORTM
headband binocular magnifier equipped with a No. 7 lens, which provides a
2.75X
magnification) were used to aid visual confirmation of embryo size and
suitable
truncation of the pericarp. lithe first cut did not remove enough of the
kernel apex, a
second cut could be made, Other low magnification devices, using the same or
similar magnifications could be used. For example, available lenses for the
Opti-
VISORTM provide magnification ranging from 1.5 to 3.5X.
Example 3: Extrusion of Embryos and Endosperms
This example describes an improvement to one embodiment of the method of
the present invention, as described in Example 1. Powered devices may be used
to
assist in the extrusion of embryos and endosperm. For example, a power chisel
such
as a WeCheer 320 power chisel, fitted with a rounded extruder device, can be
used to
reduce the force a person needs to exert to eject the embryos and endosperms.
Other
powered devices are available and can be similarly used. Preferably, the
"chisel"
portion of such a tool (or any part of the tool that might come into contact
with the
embryos) can be conveniently sterilized, for example, by insertion into a bead
sterilizer,
19
CA 3028772 2019-01-02
In one experiment, the blade of a stainless steel weighing spatula was bent
back on itself to provide an extruder device having a rounded leading edge.
After
insertion into a WeCheer 320 power chisel, a portion about 10 centimeters long
extended out from the power chisel's chuck. This assembly was used to eject
the
embryos and endosperms from individual rows of decapitated kernels. As the
extruder device (modified spatula) moved down a row of kernels, a slight
tendency
for the spatula to slide off center to the left or right was observed;
however, this
tendency could be corrected by including a small keel-like extension of the
spatula on
each outer edge.
Example 4: Mechanized Embryo Extrusion
This example describes an improvement to one embodiment of the method of
the present invention, as described in Example 1. Mechanization of the embryo
extrusion process can be achieved by use of a suitable device, such as, but
not limited
to, the device described herein and schematically diagrammed in Figure 1. This
device includes two motors. The first motor D is a stepper motor that can
rotate the
corn ear so that new rows of kernels are exposed to the two extrusion rods G,
which
apply force to squeeze the embryos and endosperms out of their pericarps.
Rods G are conveniently located on opposite sides of the ear in order to
balance the pressure applied to the ear relative to the ear's longitudinal
axis. However
a single rod can be used, or more than two rods; where multiple rods are used,
it is
preferable that they are positioned so as to evenly distribute the resulting
mechanical
pressure around the ear. The rod need not be a straight rod; in one embodiment
of the
device, a flexible "collar" encircling the circumference of the ear is used
instead of a
rigid rod. In another embodiment, multiple short rods or rollers are arranged
in a
flexible, circular configuration that can be slid along the ear's longitudinal
axis,
applying mechanical pressure to many or all rows of kernels simultaneously.
The second motor is connected to the pinion gear E connecting to a rack F so
that up and down linear motion of the ear occurs. The base of the ear is held
firmly to
a handle B by means of a screw extending from the handle down into the base of
the
ear. The narrowed middle portion of the handle is square so that it will not
rotate
unless the holder C to which it is attached is rotated by the stepper motor D.
Before insertion into the machine, the tops of the kernels are decapitated as
in
Example 1 so that the embryos and endosperms can be squeezed out. To start the
CA 3028772 2019-01-02
process, the ear is lowered until the two rods G are near the base of the ear
just below
the handle B. Then the rods are pressed against both sides of the ear and the
rack and
-
pinion assembly draws the ear upward. As this happens, the embryos and
endosperms
are removed from a couple of rows, fall downward into the collection dish H
resting
on the base I, and collect in a pile J. When the rods approach the apical end
of the
cob, the cob is withdrawn upward to its original starling position and rotated
slightly
by the stepper motor until new rows of kernels come into position.
Various degrees of automation of this machine are possible, including sensors
to automatically adjust the vertical starting and finishing positions as well
as the
rotary start and finish positions. A rack and pinion is not the only method by
which
linear motion can be obtained. Pneumatics or hydraulics may be preferred for
some
applications. Rods G can be automatically opened by a suitable mechanism. When
a
new ear is loaded, it may be preferable to raise the ear to a position high
enough to
clear the rods.
Example 5: Hydrophobic Separation of Embryos
This example describes an improvement to one embodiment of the method of
the present invention, as described in Example 1. In separation applications
the
material of interest frequently appears at the interface of dissimilar phases
(for
example, between aqueous and lipophilic solvents). Removing the material of
interest
from such an interface can pose problems, and has in the past been a manual
process
involving close contact with the extractant and the material to be extracted.
Often the
only way to successfully separate out a component is to use a material of the
same
polarity or hydrophobicity/hydrophilicity. In the case of immature corn
embryos
extruded by a method of the invention, the embryos are found at the
aqueous/air
interface. The corn embryos' surface is waxy, i, e., lipophilic or
hydrophobic, and
when an embryo cuticle is contacted with a substance of similar
hydrophobicity, the
embryo will tend to stick to the hydrophobic surface. The embryo's
hydrophobicity
reduces the surface tension of the water around it, which helps the embryo to
"float"
at the surface of the aqueous/air interface.
One approach that takes advantage of these physical characteristics would be
to touch the floating embryos with a hydrophobic material (such as hydrophobic
filter
paper, e. g., \VhatmanTM No. 1 PS paper, winch is a water-repellant phase
separating
paper impregnated with silicone.
21
CA 3028772 2019-01-02
In one example, a piece of sterile hydrophobic filter
'paper can be lowered onto an entire container of
floating embryos and pick them all up at once. In another example, a small
piece of
the hydrophobic paper can be used to successively pick up a number of embryos
and
transfer them to the next container. In a third example, either a small piece
of the
hydrophobic paper or a hydrophobic pipette tip would be used to contact and
pick up
individual embryos and then dispense them with a puff of air from the
pipettor.
Ordinary pipette tips could also be modified for such use by inserting a
pipette tip into
a short length of hydrophobic tubing (for example, silicone tubing); the
embryo could
then be picked up by hydrophobic attraction to the distal end of the
hydrophobic
tubing, and then released by dispensing a puff of air from the pipette.
Reduced
surface tension around the hydrophobic embryos helps them float on an aqueous
surface, and the floating embryos could also be transported by moving them on
the
aqueous surface (for example, by an air jet directed at the embryos). Picking
up and
dispensing of embryos can be automated using modifications of existing
devices, such
as machines designed for colony picking or for retrieving protein spots on
stained 2-D
=
protein gels.
Example 6: Further Methods of Ejecting or Extruding Embryos
The method of the present invention encompasses the use of various types of
force, or combination of forces, for separating the embryo from its seed. This
example describes further embodiments. In one basic method as described in
Example 1, mechanical positive pressure is applied to the base of a truncated
seed
(such as a corn kernel) to eject the embryo out through the truncated top of
the seed,
In another embodiment, centrifugal force can be used to eject the embryo. For
example, a corn ear (the kernels of which have previously been truncated)
could be
spun about its longitudinal axis at a speed sufficient to eject the embryos
and/or
endospenns in a radial trajectory. Spinning could be achieved by any suitable
technique, such as, but not limited to, contacting the apical end of a corn
ear with a
freely rotating cone, wherein the rotation of the ear is kept within a limited
longitudinal range, for example, by attaching the basal end of the ear to a
handle
which is then inserted in a bolder within which it can rotate. In one
exemplary
embodiment using centrifugal force, about a third of the top of each kernel on
a corn
ear was removed with a scalpel, and the ear rolled on a surface to loosen the
embryo
22
CA 3028772 2019-01-02
and endosperm within the kernels. The ear was snapped into two pieces, each
about
750 millimeters in length. Each piece was placed in a 250-millilter centrifuge
bottle
with about 100 milliliters of water. These were centrifuged 15 minutes at 5000
rpm to
eject the embryos. Examination of the ears after centrifugation showed that,
in some
portions of the ear, all the embryos had been removed by the centrifugation,
whereas
in other areas, few or no embryos were removed. The ejected material was
centrifuged and the supernatant removed to leave a slurry, which contained
intact
embryos (estimated to include about 20 percent of the total number of
embryos). In
another example, an immature ear of corn is harvested (typically between about
10 to
about 14 days post-pollination). The ear is disinfested, and under sterile
conditions
the top of each kernel is cut off. The ear is mounted on a drill bit on an
electric drill
(or a similar device) and the ear is surrounded by a large sterile collection
vessel (e.
g., a large glass beaker). The ear is spun at a rotation sufficient to eject
the immature
embryos, and the ejected tissues are collected from the sterile container.
Immature
embryos are collected, for example, by manual collection, or by rinsing the
container
with sterile tissue culture medium and recovering an enriched fraction
containing the
embryos (e. g., by sieving, by the use of a liquid density gradient, or by
other methods
to separate embryos from non-embryo tissues as described elsewhere in this
disclosure). The immature embryos (or callus derived from the immature
embryos)
can be used subsequently for transformation. Improved results using these and
other
centrifugation methods can be obtained by determining preferred centrifugation
times
and speeds by routine testing.
Another embodiment employs bulk maceration of kernels. An immature ear
of corn is harvested (typically between about 10 to about 14 days post-
pollination).
The ear is disinfested. The pericarp can be opened under sterile conditions or
the
kernels can be left intact. The kernels are removed from the cob by any
suitable
procedure, including, but not limited to, using a scalpel or other bladed
tool. The
kernels, once separated from the cob, are placed in tissue culture medium. The
kernel-medium mixture can be subjected to further tissue disruption using a
suitable
cutting device, such as, but not limited to, a blender. Immature embryos are
collected,
for example, by manual collection, or by rinsing the container with sterile
tissue
culture mediutn and recovering an enriched fraction containing the embryos (e,
g., by
sieving, by the use of a liquid density gradient, or by other methods to
separate
embryos from non-embryo tissues as described elsewhere in this disclosure).
23
CA 3028772 2019-01-02
Immature embryos (or callus derived from the immature embryos) can be used
subsequently for transformation.
In a further embodiment, fluid jets (of gases or liquids or combinations
thereof) could be used to dislodge embryos. One example of this approach is to
automatically rotate a corn ear in a stepwise or continuous (helical) manner
past a
stationary jet, collecting the ejected material containing the embryos and
further
isolating the embryos if necessary, for example, by size separation on a mesh
or
screen or the like. Where the corn ear is vertically orientated (with respect
to its
longitudinal axis), it may be preferred to rotate the ear in an upward helical
direction,
or otherwise move the ear relative to the jet so that extracted embryos tend
to wash
downward.
In yet another embodiment, linear deceleration or linear acceleration could be
employed to dislodge or eject the embryos. For example, a corn ear could be
administered a shock parallel to the ear's longitudinal axis and of sufficient
force to
eject the embryos and endosperms. A corn ear could be enclosed in a suitable
sterile,
high impact-resistant holder, which could be subjected to sudden acceleration
or
deceleration, for example, by a sharp impact (e. g., as from a mallet).
Another improvement to the method would be to facilitate ejection or
extrusion of' the embryo from the truncated seed. For example, embryos could
be
loosened or dislodged within their native position within the seed by applying
a force
to the tops of intact seeds (e. g., by applying a roller or other mesnq of
applying
pressure to the tops of rows of corn kernels in an intact ear or rolling or
pressing the
ears themselves on a surface prior to decapitating the tops of the kernels).
Embryos
may also be loosened within the seed by application of vibration, for example,
by
ultrasound. Another approach would be to remove additional non-embryo tissue,
such as additional lateral wall (pericarp) material, before embryo ejection or
extrusion. For example, a V-shaped knife or other instrument could be used to
remove some of the lateral walls of corn kernels in rows in the ear.
Example 7: Automated Embryo Isolation Using Fluid Jet Positive Pressure
This example describes a further embodiment of the present invention_ In this
example, an automated device uses fluid jet positive pressure to dislodge
embryos
from seeds. With reference to Figure 2, a robotic grasper C (preferably
capable of
motion in three dimensions by means of robot A and motor B, or by an
equivalent
24
CA 3028772 2019-01-02
means) picks up a corn ear I (by a handle D having a baffle E) in a defined
position
for a rack on the robot deck. The robot inserts the corn ear into tube H
(optionally
made of transparent material for ease of visual observation) at a starting
position
below flange F. Fluid jet positive pressure is introduced through aperture G
and the
ear is simultaneously raised (in the Y dimension) and rotated by robot A and
motor B,
preferably resulting in each kernel being struck by the fluid jet, causing the
embryo
and endosperm to be dislodged. The fluid passing through aperture G can be at
least
one gas, at least one liquid or any combination thereof. The fluid jet can
exert force
continuously or non-continuously, for example, as in pulses. As the embryos
and
endospernis are dislodged by fluid jet positive pressure from aperture G, they
fall
down to the shaking screen J, which retains the endosperms while permitting
the
embryos to fall through to the collecting surface K (for example, sterile
cheesecloth)
below. Excess fluid can be optionally collected in waste or recycling
receptacle L.
After completion of the embryo removal process for each corn ear, the interior
of the
tube can be briefly washed down manually or by automated jet above or below
the
flange F.
Example 8: Methods of Processing Crude Embryo Preparations
The embryo preparations obtained by methods such as those described in
Examples 1 through 7 may include both intact embryos and partial embryos,
which
may be accompanied by non-embryo tissues, such as endosperm and glumes. Some
applications may not require further treatment or separation steps, for
example, in a
mass transformation of such a "crude" embryo preparation where embryos (intact
or
partial) need not be separated from non-embryo tissue. For example, callus
derived
from either intact or partial immature corn embryos can be used for
transformation,
regeneration, and production of fertile, transgenic plants. Thus, both intact
and partial
embryos may serve as transformable explants and need not be separated from
each
other. However, in other cases it may be desirable to further purify embryos
from a
crude embryo preparation.
Procedures wherein some difficulties may be encountered in processing crude
embryo preparations include: (1) rinsing away of non-embryo tissue (e. g.,
cell
debris, starch grains, undesirable proteins), (2) efficiently removing excess
liquid
from embryos after extrusion or rinsing using liquid, and (3) adding liquid
with
minimal turbulence so that the embryos float and do not become submerged.
CA 3028772 2019-01-02
A porous material is useful for separating non-embryo tissue from embryos.
Any suitable porous material can be employed, preferably having a mesh or hole
size
small enough to retain embryos but let smaller, non-embryo tissues or debris
pass
through, and capable of being sterilized (e. g., by autoclaving, heat,
irradiation, or
chemical sterilization). Suitability of materials is easily judged or tested
by simple
experimentation by one skilled in the art. Examples of suitable materials
include
cheesecloth or other woven material, and other meshes or screens. In some
embodiments, perforated solid materials can be used, including perforated
ceramics,
polymers, metals, or glasses (for example, in the form of a Bticluier or
similar
separatory funnel). Cheesecloth of appropriate gauge, for example, has a mesh
size
small enough to retain embryos but allows smaller debris to pass through, and
is
autoclavable. Cheesecloth can be attached to a frame or collar (for example,
the
frame holding embryo collecting surface K in Figure 2 and described in Example
7)
to allow the cheesecloth and all the retained embryos to be simultaneously
submerged
for easy rinsing. For example, cheesecloth can easily be attached to the frame
by
means of an elastic band or the like (e. g., silicone tubing); such frames are
easily
manufactured, for example, from a beaker or graduated cylinder made of
autoclavable
material (e. g., polypropylene, polymethylpentene, polycarbonate, or
autoclavable
glass) cut into sections. Cheesecloth has strong capillarity, allowing liquid
to be
efficiently pulled away from the embryos, thus exposing their waxy epidermis
to air
prior to flotation. In the flotation step, the cheesecloth is simply submerged
in
aqueous liquid, allowing the embryos to float off.
Example 9: Substantial Isolation of Embryos Using a Fluid Jet
This example describes a further embodiment of the present invention. In this
example, multiple embryos were dislodged from seeds by fluid jet positive
pressure.
In the simplest example, a 200-microliter pipette tip was attached to a
vertical
sink nozzle with Parafilm1). When the tap water was turned on a jet emerges
from the
pipette idtp with considerable force. The tap water pressure was estimated to
be about
60 pounds per square inch. This fluid (liquid) jet was trained on an immature
corn ear
(contained in a beaker) wherein the kernels had been decapitated as described
in
Example 1. As the jet stuck each kernel, the endosperm and embryo were
ejected,
and collected in the beaker. Since the endosperm at this stage is a relatively
soft
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tissue it was fragmented into many smaller pieces by the jet, whereas the
embryos
appeared to remain intact
The endosperm and embryo tissue dislodged by the jet was poured directly
onto a No. 60 cheesecloth (other suitable porous material, such as hydrophilic
mesh of
the appropriate mesh size, could be substituted). Different "grades" of
cheesecloth
are available (for example, grades 10, 20,30, 40, 50, 60, 70, 80, and 90,
where the
mesh openings decrease with higher grades), and the grade or mesh size
appropriate
to the average size and shape of a given type of embryo is easily selected by
simple
experimentation. The embryos and larger fragments of the endosperm were
retained
on the upper surface of the cheesecloth. Prior to the next step, the
cheesecloth was
allowed to partially dry by wicking away excess liquid. This pulled liquid
away from
the tissues and exposed the surfaces of the embryos to air. When the
cheesecloth was
lowered into aqueous liquid, the embryos floated because their waxy epidermis
did
not rewet
In a simple set up, the cheesecloth (or other suitable porous material) can be
manually stretched or held over a receptacle or waste container as the liquid
holding
the crude embryo preparations is poured through the cheesecloth. For sterile
work,
the cheesecloth can be attached to rigid frames, which can be autoclaved
before use.
Snap-together sieves with handles, such as those available in kitchen supply
stores,
could also be used in The method.
Example 10: Devices for Embryo Extraction Using a Fluid Jet
This example describes various embodiments of an apparatus for mechanically
preparing multiple corn embryos suitable for tissue culture.
One embodiment includes an apparatus for preparing multiple corn embryos
using a fluid jet, generally similar to the device depicted in Figure 2. A
transparent,
open-ended cylinder was made by cutting the ends off a 1-liter autoclavable
TM
polymethylpentene (PM?) graduated cylinder. A pipette tip (1250-microliter
Gilson
Distritip, tapered to avoid backpressure build-up) was secured to the side of
the
cylinder and served as an aperture for guiding a fluid stream as a jet through
a hole
made in the cylinder's wall. Fluid (in this case, water) was fed through the
pipette tip
from PharMede high pressure autoclavable peristaltic pump tubing; the water
was
delivered from a laboratory sink tap, but could be an aqueous fluid delivered
from a
pump or other source. Using a pump capable of delivering a sterile fluid is
preferable
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when, for example, sterile culture medium or a sterile salt solution is found
to be
superior to water as a liquid for substantial isolation of embryos. An example
of a
suitable pump is a Masterflex TM pump with the high pressure L/S pump head,
which can
deliver sterile liquid at up to 100 psi when used with high pressure tubing.
A corn ear with previously decapitated kernels was manually positioned
within the cylinder. Once the ear was positioned appropriately within the
cylinder,
each kernel was subjected to positive pressure from the water jet. This
resulted in the
embryos and non-embryo tissues being extruded from the kernels. Examination of
the ear after this treatment indicated efficient removal of the embryos from
the
kernels. The extruded material was washed down the cylinder's interior walls
to an
embryo collector positioned beneath the cylinder. The embryo collector
included: (1)
a coarse plastic screen (onto which larger debris was trapped), heat-fused to
the cut-
off top of a Tri-Pourrm plastic beaker and stacked above (2) a finer screen
(Grade 60
cheesecloth, onto which the extruded embryos were trapped), secured with an
elastic
band to the cut-off top of a second Tri-Pour'm plastic beaker and stacked
above (3) a
waste collection beaker or other container (in which the fine debris, non-
embryo
tissues, and waste liquid was collected).
Modifications to these and similar embodiments are easily made by one versed
in the art For example, with regard to positioning the corn ear or seed for
application
of the fluid jet, the ear could be held manually in place, or preferably,
mounted
securely within the cylinder by a movable support capable of moving the ear in
three
dimensions. For example, the ear could be mounted to a threaded metal or
polymer
rod, such as a polypropylene rod, which could be used to move the ear along
its
longitudinal axis as well as to rotate the ear). Another example of a mounting
mechanism is depicted in Figure 3, which illustrates a magnetic "handle" by
which an
ear can be secured to a robot arm.
In other embodiments, however, the corn ear or ears need not be individually
secured to a holder but can be freely movable so as to allow multiple kernels
to be
contacted by the force used to remove the embryos from the kernels. For
example, at
least one ear, or multiple ears, can be borne on or held between at least one
support,
such as, but not limited to, at least one plane, frame, grid, screen, mesh,
platform,
roller, guide wire or rod, and belt, wheel, or roller conveyor. Such a support
could be
movable or could cause the ear or ears to move, for example, by vibration,
rolling
motion, gravity, or other mechanisms, Substantially isolated embryos could
pass
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through the platform itself if the platform was porous (e. g., made of mesh).
The ear
or ear can also be floated on a fluid in a manner allowing each ear to rotate
or
otherwise move freely while afloat. The fluid, such as a liquid containing the
substantially isolated embryos, could be continually drained off, optionally
through a
filtering or sedimenting device, or collected for centrifugation.
Devices for obtaining motion along the longitudinal axis of a corn ear
include,
but are not limited to, ball screw-driven slides or belt-driven slides, such
as those
commercially available from various manufacturers such as Techno, Inc. (techno-
isel.com). To obtain rotary motion for rotating a corn ear, a stepper motor
can be
used, for example, a stepper motor attached to a slide plate. Rotary motion
can also
be provided by rolling devices, for example, by parallel round or tubular
rollers
between which the corn ear is held and rotated.
The shape of the fluid jet can be advantageously modified according to the
desired application. For example, a narrow column-shaped jet of uniform
diameter is
useful for removal of embryos from one seed at a time. Where it is desirable
to
increase the rate at which embryos are substantially isolated, multiple
embryos can be
simultaneously removed from their seed by a fluid jet; this can be achieved,
for
example, by using at least one single fluid jet that covers a larger area, or
by using
multiple jets simultaneously. In one embodiment, multiple jets, such as
multiple
parallel, narrow, column-shaped jets (for example, produced by multiple
nozzles
similar to that used in Example 9 and optionally connected to each other by a
manifold) are used to direct fluid jet positive pressure on multiple seeds to
substantially isolate their embryos substantially simultaneously. Automation
of these
and other devices can further include optical or mass sensors to aid in
positioning the
ear and fluid jet relative to each other.
In another embodiment, at least one fluid jet that covers a larger area (for
example, wherein the fluid jet simultaneously impacts multiple kernels, or
multiple
rows of kernels on a corn ear) can be used. The dimensions of such a jet
preferably
allow the jet to enter the kernels and wash out the embryo. Typically, corn
embryos
used in genetic experiments are immature and generally in the size range of
about 1.8
to about 2.2 millimeters in length; the kernels holding these immature embryos
are
generally in the size range of between about 4 and about 5 millimeters in
width. For
embryos of this size, an appropriate fluid jet can be, for example, between
about 0.5
to about 1 millimeter in width.
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My suitable means for producing such a larger fluid jet may be used, such as,
but not limited to nozzles that generate non-colnremr fluid jets. Examples of
suitable
nozzles include, but are not limited to, nozzles that generate a flat spray
pattern and
nozzles that generate a fan- or a cone-shaped spray pattern. In one example, a
commercially available flat spray nozzle (number 23990-1/4-04, Spraying
Systems
TM TM
CO. Dillburg, PA) was used with a Masterflex L/S pump (model
77250-62) to pump
liquid at 1 liter per minute and 30 psi; embryos were excised from a corn ear
under
these conditions. Another example of a preferred nozzle is a nozzle that
generates a
fluid jet in the form of a flat "sheet" of fluid, such as is depicted in
Figure 4. Such a
nozzle preferably is capable of generating a uniform, flat fluid jet that
maintains a
coherent, uniform sheet-like flow for at least a distance sufficient to allow
the flow to
contact more than one seed (and preferably several seeds) at the same time.
The
novel nozzle depicted in Figure 4 is designed to generate a uniform, flat
sheet-like jet
that is about 0.5 to about 1 millimeter in thickness, greater than about 20
millimeters
in width, and mainteins the sheet-like flow over a distance of about 20 to
about 25
millimeters from the nozzle's aperture. This latter distance permits the jet
to be
moved along the rows of kernels with minimal adjustment needed for differences
in
distance between the surface of the kernels and the nozzle's aperture.
Regardless of the area or shape of the jet or spray pattern generated by the
nozzle or aperture through which the liquid flows, nozzles or apertures are
preferably
used with flow rates and pressures sufficient to generate enough fluid force
to
dislodge the embryo from its seed, without damage to the embryo. In some
embodiments, it is preferable to use a lower flow rate and possibly a higher
pressure,
to minimize consumption of fluid (such as medium) as well as to minimize the
waste
generated.
Example 11: Using a Gas Jet to Substantially Isolate Embryos
This example describes farther embodiments of methods and devices for
mechanically preparing multiple corn embryos suitable for genetic
transformation or
tissue culture. As described in Example 6, gas jets can also be used for the
substantial isolation of multiple embryos. An apparatus similar to that
described in
Example 10 was modified for use with gas. A 1-milliliter pipette tip
(catalogue
number TN130ORS, Marsh Bio Products) was secured to the side of the cylinder
and
served as an aperture for guiding a stream of air as a jet through a hole made
in the
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cylinder's wall. Air was supplied from a compressor pressurized to between
about 60
to about 100 psi. An air valve for convenience was positioned in line between
the
compressor and the pipette tip. The air jet emerging from this pipette tip was
used to
dislodge the embryos from a prepared corn ear. Examination of the kernels
after they
had been subjected to the air jet showed that the thick pericarp remained in
place and
surrounded by papery gltunes, and the pericarp contents (embryo and endosperm)
had
been removed. Examination of the tissue retained by the grade 60 cheesecloth
showed that this included dislodged embryos as well as some glumes dislodged
by the
high-pressure air jet. The glumes of corn have a waxy surface like the embryos
and
also float following the flotation procedure. Using lower air pressures can
reduce
glume contamination.
Example 12: Substantial Isolation of Embryos Using Other Fluid Forces
This example describes further embodiments of methods and devices for
mechanically preparing multiple corn embryos suitable for genetic
transformation or
tissue culture. Forces exerted by fluids, other than positive fluid pressure
from a fluid
jet, can be used to substantially isolate embryos. In one experiment, the tops
of
kernels were removed from a com ear, which was placed inside a bottle
containing
sterile distilled water and shaken vigorously by hand. This resulted in the
substantial
isolation of 90 out of the ear's 200 embryos. Another experiment repeated the
preceding procedure except that the shaking was carried out in a mechanical
paint
shaker. In this experiment, 56 embryos were substantially isolated out of the
ear's
190 embryos. In a third experiment, a similar procedure was carried out,
except that
the corn ear was pre-soaked in 211 medium, and the shaking was carried out in
a paint
shaker. In this experiment, 109 embryos were substantially isolated out of the
ear's
210 embryos. In these cases, non-jet fluid force from movement of the liquid
around
the corn ear resulted in the substantial isolatation of the embryos; the fluid
force could
include fluid turbulent flow, fluid laminar flow, shear from fluid flow,
negative fluid
pressure (for example, resulting cavitation), or combinations thereof. Forces
can also
include forces generated by acoustic techniques, such as by an acoustic wave
or
waves (pulsed or continuous) in either gas or fluid phase.
Preceding examples (including Examples 9¨ 11) described use of a fluid jet
to remove embryos from an immature ear. During these procedures, it was
observed
that the fluid jet generally also caused at least part of the endosperm to be
released
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=
from the kernel. The endosperm tissue was observed to be softer and more
friable
than the embryos, and tended to disintegrate to varying degrees (in contrast
to the
embryos, which tended to remain intact). It is possible that the endosperms
disintegrate upon exposure to shear caused by the fluid jet. This shear is
believed to
be non-uniform, resulting in the variability in disintegration observed;
nonetheless, a
large proportion of the endosperm material that was sufficiently disintegrated
to pass
through the cheesecloth, leaving a retentate made up of a semi-pure
preparation of
embryos.
When a low-pressure jet from an ordinary laboratory squirt bottle was directed
at the cheesecloth retentate, more of the remaining endosperm tissue was
disintegrated
further and washed through the cheesecloth, leaving behind a relatively more
pure
preparation of embryos. Thus it is reasonable to predict that if the retentate
is
imifonnly exposed to a shear force of the correct intensity, all or
substantially all of
the remaining endosperm should disintegrate and pass through the cheesecloth.
Such
a shear force could be generated by any suitable means, such as, but not
limited to, a
single jet, multiple jets, a sheet-like or curtain-like jet, rapidly moving
jets, and
acceleration or deceleration of the endosperms. Additionally, if the jet used
to
initially release the kernel contents is designed to expose a higher
proportion of the
endosperms to shear during ejection, an initial higher purity embryo
preparation could
be obtained.
One non-limited embodiment of applying shear to further purify embryos
follows. Once the embryos and partially disintegrated endosperms are released
from
a cob, the remainder of the endosperm can be rapidly fragmented by fluid flow,
for
example, from a spray nozzle, that strikes the endosperm uniformly and
simultaneously. One suitable type of nozzle is a full cone nozzle. Full cone
nozzles
generate a spray pattern completely filled with drops. An internal vane within
the
nozzle imparts controlled turbulence to the liquid prior exiting to the
orifice, allowing
formation of the spray pattern. Commercially available nozzles have spray
patterns
that are round, square, or oval. An example of a suitable full cone nozzle is
known as
"UniJet TU Spray Nozzle, Standard Spray, Small Capacity" (part number TG-
SS0.3).
Example 13: Combination Devices
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This example describes several additional embodiments of the method of the
invention, which use a combination of forces to substantially isolate multiple
embryos
from seeds.
Figure 5 illustrates a device using a larger fluid jet (as described in
Example
10). This device includes a nozzle for generating a fluid flow such as a
larger fluid jet
(for example a flat fluid jet), and, optionally, a suction head, or component
for
applying negative fluid pressure (e. g., by vacuum or suction), for dislodging
embryos
and/or for collecting the dislodged embryos. Figure 5 (top) depicts a cross-
sectional
view of an example of such a device, showing how the nozzle, optional suction
head,
and corn ear can be positioned relative to each other. The corn ear, nozzle,
and
optional suction head can be moved relative to each other; for example, the
corn ear
may be stationary while the nozzle and optional suction head are moved, or the
nozzle
and suction head may be stationary while the corn ear is moved. Figure 5
(bottom)
schematically depicts a corn ear positioned in the device, and shows the
nozzle
positioned to generate a flat fluid jet wherein the jet impacts multiple
kernels in a row.
Figure 6 depicts an embodiment of a suitable suction head or component for
applying negative fluid pressure (e. g., by vacuum or suction), such as is
optionally
used in the device of Figure 5, and which can also be used on its own to
substantially
isolate embryos. The suction head can include one or more apertures through
which
negative fluid pressure can be applied. The suction head can also include a
means for
dispensing fluid (such as gas or liquid, e. g., water or medium), for example,
multiple
apertures in the suction head. For use with corn, the suction head is
preferably shaped
to follow the contours of a typical corn ear, and is preferably capable of
entrapping
embryos from multiple kernels or from multiple rows of kernels. It is
envisioned that
the suction head can be manufactured of a rigid material (such as stainless
steel or
other metals), or of a flexible material to allow easier conformation of the
suction
head to the contours of a corn ear, or of combinations thereof. Embryos can be
substantially isolated by any combination of mechanical positive pressure
(exerted,
for example, by a leading edge of the suction head), negative fluid pressure
(e, g.,
suction or vacuum), and fluid force (such as, but not limited to, positive
pressure from
a fluid jet, fluid turbulent flow, and fluid laminar flow entrapping material
from the
interior of the kernel)
Devices for applying force for substantially isolating embryos, such as are
described in Examples 1, 3, 4, 6, 7, 9, 10, and the present example
(including, but not
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limited to the devices illustrated in Figures 5 and 6) can be moved relative
to the corn
ear. The ear may be stationary, or the device may be stationary, or both can
be
moved. Because corn seed typically occurs in relatively uniform rows arranged
parallel to the longitudinal axis of the corn ear, the device is typically
moved (relative
to the ear) so that the device passes parallel to the longitudinal axis of the
corn ear and
following a row or multiple rows of kernels. However the motion of such
devices
relative to the ear can follow the circumference of the ear, or can be random,
or can be
any combination of suitable motions.
Figure 7A through 7C depict different views of an embodiment of a device
that uses a combination of forces to substantially isolate multiple embryos
from seed
(in this example, corn). This device includes a head with a leading edge
capable of
applying a predefined amount of mechanical pressure to the base of kernels
that
previously have had the pericarp opened or truncated, so that the embryos are
extruded from the kernels in a manner similar to those described in Examples
1, 3,
and 4. The device further includes a component for applying negative fluid
pressure
(e. g., by vacuum or suction) for dislodging embryos and/or for collecting the
dislodged embryos. The extruded embryos (and accompanying non-embryo tissues)
are thus separated from the corn ear and can be collected by application of
negative
fluid pressure. The collected embryos and non-embryo tissues can be further
separated, if desired, by suitable means, such as by size-separation,
hydrophobic
separation, or differential centrifugation. A variation of this device could
include a
means for dispensing fluid (such as liquid, e. g., water or medium), for
example,
multiple apertures in the suction head.
The embryo extraction devices depicted in Figures 5, 6, and 7 are described as
illustrative examples that are not intended to be limiting. These and other
such
devices can include additional components, for example, means for separating
the
embryos from non-embryo tissues or from fluids used in the substantially
isolation
process.
Example 13: VIABILITY DATA
The multiple monocot embryos provided by use of the methods and devices of
the present invention are most preferably embryos suitable for genetic
transformation
or tissue culture application such as transformation and regeneration of
plants. This
example further illustrates the utility of methods of the invention in
providing
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multiple monocot embryos that are viable and suitable for genetic
transformation or
tissue culture. In this example, the quality of immature corn embryos obtained
by
different excision methods was compared in their response to transformation by
Agrobacterium tumefaciens.
For transformation, a plasmid containing left and right border sequences, a
gene for glyphosate resistance for selection, and a reporter gene (g),
encoding green
fluorescent protein) was used. Agrobacterium containing this plasmid was
streaked
from a frozen glycerol stock onto an LB plate and grown for 3 days in a 28
degree
Celsius incubator. A seed culture was prepared by inoculating three colonies
from the
LB plate into 25 milliliters of LB broth, which was incubated 15 hours at 27
degrees
Celsius with shaking (200 spin). This seed culture (10 milliliters) was
diluted with 40
milliliters of fresh LB broth and grown for 6 hours at 200 rpm at 27 degrees.
Agrobacterium was centrifuged for 10 minutes, and the pellet resuspended at an
optical density of 0.2 at 660 nanometers in AB minimal induction media. This
was
incubated 15 hours at 27 degrees Celsius with shaking (200 rpm). The
Agrobacterium
culture was centrifuged for 10 minutes and the pellet washed withl 0
milliliters Lynx
1040 and resuspended in 5 milliliters inoculation medium. The optical density
was
adjusted to 1.0 and used for inoculation.
Four experiments (designated A, B, C, and D respectively) were performed..
Each experiment compared embryos obtained by manual excision to embryos
obtained by a method of the present invention: excision by a liquid jet
(experiments
A, B, and D) or excision by a gas jet (experiment C). The liquid jet in
experiments A
and B used ordinary tap water and a nozzle made of a pipette tip. Experiment C
tested a gas jet using air from a compressed-air pump and a nozzle made of a
pipette
tip. The liquid jet in experiment D used Vi MSPL medium as the liquid and a
solid
stream nozzle with an equivalent orifice diameter of 0.020 inches (catalogue
number
TP000050-SS, Agricultural Division of Spraying Systems CO.Tm, Dillburg, PA).
Corn ears were harvested twelve days after pollination and sterilized by
soaking in a 1-liter bottle of 80% ethanol for 3 minutes. Embryos were
manually
excised by cutting off the top third of the kernel with a scalpel and removing
the
embryo from the kernel using a narrow spatula. The collected embryos were
excised
into 1 milliliter of V2 MSPL medium in a single microcentrifuge (Eppendorf)
tube.
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The medium was removed and replaced with 1 milliliter of Agrobacterium
tumefaciens prepared as described below
Embryos were also substantially isolated using a fluid (liquid or gas) jet,
following procedures similar to those described in Examples 10 and 11. The
fluid jet
was used to excise the remaining embryos on the ears after removing the top
third of
the kernel with a scalpel. The ear was positioned so that the fluid jet was
aimed into
individual cut kernels in succession to dislodge both the embryo and non-
embryo
tissue (endosperm). The kernel contents removed from the ear were passed
through a
coarse screen to remove large pieces of endosperm, and the embryos were
collected
on sterile cheesecloth. Embryos were transferred using a small spatula from
the
cheesecloth into a microcentrifuge tube containing 1 milliliter of V2 MSPL
medium.
After all of the embryos were collected, itS MSPL medium was removed and
replaced
by 1 milliliter of Agrobacteriwn tumefaciens inoculant.
Embryos prepared by the various excision methods were subjected to the same
inoculation, selection, and regeneration procedures. Suitable procedures,
including
descriptions of media and reagents, for transformation of plants using
glyphosate
selection and GFP as a reporter have been disclosed in United State Patent
Application Publication Number 2004/0244075 to Cal et al.
Embryos were inoculated with 1.0 milliliters of Agrobacterium for 5 minutes.
The contents of the microcentrifuge tube were poured onto a plate of co-
culture
medium, and co-cultured for 18 hours at 23 degrees Celsius. Embryos were
transferred next to induction MS medium, and cultured at 30 degrees Celsius
for 13
days. Celli derived from the transformation were cultured at 27 degrees
Celsius for
11 days prior to regeneration. At this time, GFP positive sectors were counted
using a
fluorescence microscope. For regeneration, calli derived from each embryo were
individually transferred to MS/6 BA medium and cultured in a light room for 7
days,
after which each greening callus was transferred to MSOD medium and returned
to
the light room for 17 additional days. Resulting shoots were transferred to
Phytatrays
containing regeneration medium (consisting of 2.165 g MS basal salts, 5
milliliters
100X MS vitamins, and 20 grams sucrose made up to 1 liter in water and
autoclaved,
pH adjusted with KOH to 5.8, solidified by autoclaving with 3 g Phytagel, and
with
0.75 milliliters of 1 milligram per milliliter indole-3-butyric acid, 0.5
milliliters of 1
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milligram per milliliter 1-naphthaleneacetic acid, and 0.2 milliliters 0,5
molar
glyphosate added). After about 3 weeks, transgenic plants were hardened off by
transplanting rooted shoots in peat pots containing soil mix and grown at 26
degrees
Celsius.
The results of these experiments axe summarized in Table 1. The number of
embryos that were transformable is estimated from the number of GFP-positive
embryos. Overall transformation and regeneration frequency is given as the
percentage of GFP-positive plants regenerated from the inoculated embryos.
These
results demonstrate that various methods and devices of the present invention
are
useful for providing multiple monocot embryos suitable for genetic
transformation or
tissue culture,
Table 1
experiment excision number of number
transformation number of transformation/
method embryos of GFP- frequency plants to
regeneration
inoculated positive soil frequency
embryos
A manual 56 23 41% 6 11%
liquid jet 44 8 18% 3 6.8%
manual 22 11 50% 6 27%
liquid jet 23 4 17% 1 4%
manual 33 27 82% n/a n/a
gas jet 61 19 31% n/a n/a
manual 36 17 47% n/a n/a
liquid jet 166 51 31% n/a
n/a: data not available
All of the materials and methods disclosed and claimed herein can be made
and used, as instructed by the above disclosure, and without undue
experimentation,
by a person of ordinary skill in the art. Although the materials and methods
of this
invention have been described in terms of preferred embodiments and
illustrative
examples, it will be apparent to those of skill in the art that variations may
be applied
to the materials and methods described herein. The scope of the claims should
not be
limited by the preferred embodiments set forth herein but should be given the
broadest
interpretation consistent with the description as a whole.
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