Note: Descriptions are shown in the official language in which they were submitted.
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MICROPROJECTILE BOMBARDMENT TRANSFORMATION
OF BRASSICA
FIELD OF THE INVENTION
The field of the invention relates to the genetic engineering of plants,
particularly methods for genetically transforming Brassica plants.
BACKGROUND OF THE INVENTION
Brassica species are used as a source of vegetable oil, animal feeds,
vegetables and condiments. Brassica plants that are used for vegetable
production
include cabbage, cauliflower, broccoli, kale, kohlrabi, leaf mustard and
rutabaga.
Seeds of B. hirta are used to produce the popular American condiment, yellow
mustard. However, on a world-wide basis, the most economically important use
of
Brassica species is for the production of seed-derived, vegetable oils. The
predominant Brassica species grown for oil production is B. napus, followed by
B.
juncea and B. rapa. Seeds of B. napus, B. juncea and B. rapa are referred to
as
rapeseed. Brassica species that are grown primarily for oil production are
often
called oilseed rape. In North America, canola, a type of oilseed rape that has
been
selected for low levels of erucic acid and glucosinolates in seeds, is the
predominant Brassica piant grown for the production of vegetable oil for human
consumption. While low-erucic-acid rapeseed oils, such as canola oil, may be
favored for human consumption, high-erucic-acid rapeseed oils are desirable
for a
variety of industrial applications including the production of cosmetics,
lubricants,
plasticizers and surfactants.
Because of the agricultural and industrial importance of plants from the
genus Brassica, plant breeders are working to develop new varieties with
improved
agronomic characteristics. While traditional breeding approaches are
important,
significant improvements in cultivated Brassica varieties have been made
recently
through ihe introduction of recombinant DNA into the Brassica genome by
genetic
transformation methods. A number of genetically modified Brassica varieties
have
already reached farmers' fields in North America. Transgenic canola varieties,
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genetically modified for resistance to herbicides, have rapidly gained favor
with
agricultural producers across the canola-growing regions of the United States
and
Canada. The phenomenal success of the transgenic canola varieties in North
America has led to acceleration in the development of new transgenic varieties
of
canola. Novel, recombinant DNA-based strategies for incorporating new traits,
such
as disease and insect resistance, modified seed oil composition and modified
seed
protein composition, are being developed for canola and other Brassica
species.
All of these strategies depend on genetic transformation methods to introduce
the
recombinant DNA into the genomes of Brassica plants.
Currently, the most favored methods for transforming Brassica species
involve the use of Agrobacterium. While the Agrobacterium-based transformation
methods provide a reliable means for introducing foreign DNA into plants,
there are
a number of disadvantages to methods of plant transformation that involve the
use
of Agrobacterium. First, an undesired consequence of all Agrobacterium-based
methods is the introduction of at least one T-DNA border into the genome of
the
recipient plant. While the T-DNA border is an essential element of the genetic
mechanism by which Agrobacterium transfers DNA to a plant cell, the T-DNA
border is not essential for the expression of foreign genes in the recipient
plant.
Additionally, the accumulation of multiple T-DNA borders throughout the genome
of
a plant may have deleterious effects on the plant or its progeny. Second, the
co-
cultivation of plant tissues with Agrobacterium may slow the regeneration of a
transformed plant from a transformed cell. After the co-cultivation phase,
Agrobacterium must be eiiminated from cultures of the plant tissues. High
levels of
bactericidal agents must be applied to the plant cultures to kill the
Agrobacterium.
While the levels of bactericidal agents applied to the cultures are generally
not
lethal to the piant tissues, the presence of the bactericidal agents in the
cultures
may negatively impact piant growth and thus, slow the regeneration of
transformed
plants. Third, prior to DNA transfer to a plant, natural genetic processes
might
occur in Agrobacterium such as genetic recombination and DNA rearrangements
that may have undesired effects on the DNA fragment that is transferred to the
plant. Such undesired effects may alter or eliminate the intended genetic
function
of the introduced DNA fragment.
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Efficient Brassica transformation methods that do not involve the use of
Agrobacterium are desired. US 6,051,756, US 6,495,741, US 2003/0093840 and
US 2004/0045056 describe the transformation of seediing hypocotyls by particle
bombardment. US 6,297,056 describes the transformation of cotyledonary
petioles. US 6,515,206 and US 2003/0200568 describe the use of transformation
of plastids in true leaves. Chen and Beversdorf Theor. Appl. Genet. 88: 187-
192
(1994) describe a biolistic transformation procedure of microspore-derived
hypocotyls involving DNA imbibition. Fukuoka et al. Plant Cell Reports 17: 323-
328
(1998) describe biolistic transformation of fresh microspores. Finally, Nehlin
et al.,
Plant Physiol. Vol. 156: 175-183 (2000) describe transient biolistic
transformation of
pre-incubated microspores, but no stable transgenics were reported.
To meet the increasing demands of agriculture in the world today, the pace
of development of new transgenic varieties of canola and other Brassica
species
must be accelerated. Increasing the pace of Brassica variety development
depends on the availability of reliable and efficient methods for the
transformation
and regeneration of transformed Brassica plants.
SUMMARY OF THE INVENTION
Methods are provided for producing transgenic Brassica plants. The
methods comprise introducing DNA constructs by microprojectile bombardment.
The introduced DNA constructs can encode proteins or can suppress endogenous
genes. The methods find use in agriculture, particularly in the development of
improved varieties of Brassica plants through the incorporation of desirable
agronomic traits. The methods involve introducing a DNA construct by
microprojectile bombardment into a Brassica cell that is capable of
regenerating
into a stably transformed Brassica plant and regenerating such a Brassica
plant
from the cell.
An aspect of the invention is to provide a method of producing a transformed
Brassica cell by particle bombardment, comprising: (a) culturing a pre-
incubated
microspore-derived explant comprising a cell under a condition of plasmolysis
for a
period of about half an hour to about 4 hours prior to bombardment; (b)
introducing
a DNA construct by microprojectile bombardment into an exposed cell on a
surface
of the pre-incubated microspore-derived explant, wherein the explant is under
the
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condition of plasmolysis; and (c) continuing to culture the bombarded pre-
incubated
microspore-derived explant under the condition of plasmolysis for a period of
about
4 hours to about 20 hours, to produce a transformed Brassica cell. A pre-
incubated
microspore-derived explant is a microspore or any tissue derived from the
microspore (for example a microspore-derived embryo or a microspore-derived
hypocotyl) that has been cultured for a period of time of between 1 day and 30
days
prior to bombardment. The condition of plasmolysis can be selected from the
group consisting of (a) culturing the explant on osmotic medium and (b)
culturing
the explant on wetted filter paper. The explant can be a pre-incubated
microspore,
a pre-incubated microspore-derived embryo, or a pre-incubated microspore-
derived
hypocotyl. The method can further comprise the steps of regenerating a
transformed plant from the transformed cell, comprising: (a) culturing said
microspore-derived explant on a regeneration medium to produce a regenerated
embryo or tissue; and (b) regenerating a fertilestably transformed Brassica
plant
from said embryo or tissue
An aspect of the present invention is to provide a method for producing a
stably transformed Brassica plant, comprising: (a) introducing a DNA construct
by
microprojectile bombardment into a pre-incubated explant, which may be a
microspore, or a microspore-derived embryo or portion of a microspore-derived
embryo; (b) culturing the pre-incubated explant to produce an embryo or
tissue;
and (c) regenerating a stably transformed Brassica plant from the embryo or
tissue.
In step (a), a pre-incubated microspore can be produced by culturing a
microspore
in a culture medium for a period of about two to ten days prior to
bombardment.
The period can be from about four to eight days, or from seven to eight days.
The
method can further comprise a step of inducing plasmolysis of the pre-
incubated
microspore prior to, during and after bombardment. For example, plasmolysis
can
be induced by (a) culturing the pre-incubated microspore on osmotic medium
prior
to, during and after bombardment, or (b) culturing the pre-incubated
microspores
on wetted filter paper prior to, during and after bombardment. The osmotic
medium
can comprise between about 17 and 19% sucrose and between about 0.8 and
1.6% Phytagel' m agar (w/v). The pre-incubated microspore can be cultured on
osmotic medium for about between half an hour and four hours prior to
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bombardment and for about between four hours and twenty hours after
bombardment.
The method can further comprise a seiection step after bombardment
comprising culturing the bombarded pre-incubated microspore on a medium
comprising a selection agent against a gene encoded by the DNA construct. For
example, the selection agent can be selected from the group consisting of
kanamycin, G418 and glyphosate. The concentration of G418 can be between
about 5 and 10 mg/I. The concentration of glyphosate in the medium can be
between about 0.1 mM and 0.2 mM. The method may further comprise a step of
orientating the pre-incubated microspore during bombardment so that a surface
of
the microspore is exposed during the bombardment. The method may further
comprise a step of collecting the pre-incubated microspore such that it is
viable and
embryogenic prior to bombardment. The step of collecting the pre-incubated
microspore can be a filtration step or a step of Percoli gradient
centrifugation
(Percofl concentration is 35-45%). The filtration step can be done using a
sieve
having a pore size of about 15 to 48 pm.
In step (a), of the method, the microprojectile bombardment can be
conducted using bombardment factors comprising about 12.5 ng to 5 pg of said
DNA construct, about 15 pg to 100 pg gold particles per shot at the size of
0.4
micron to 0.6 micron, about 2.5 M CaC12 and a 650 to 900 psi rupture disk. The
step of regenerating a stably transformed plant can comprise culturing the
bombarded pre-incubated microspore on a first liquid selection medium for a
first
period of time, a second liquid selection medium for a second period of time,
and
then transferring the resistant embryo or tissue derived from the pre-
incubated
microspore to solid medium for a third period of time. The first period of
time can
be about 7 days and the pre-incubated microspore can be cultured in darkness.
The pre-incubated microspore, or tissue or embryo derived from the pre-
incubated
microspore, can be cultured in the second liquid selection medium for
approximately 14 days in dim light of approximately 240 foot candles or 2,583
Lux.
The second liquid medium can be liquid NLN-6.5S and further comprise growth
regulators. The growth regulators in the second liquid selection medium can
comprise 0.5 mg/L NAA and 0.05 mg/1 BAP. Further, the first, the second, or
both
the first and the second liquid selection media can comprise G418 or
glyphosate.
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Further, the solid medium may comprise growth regulators to induce
regeneration,
and optionally further comprise a selection agent against a gene encoded by
the
DNA construct. The solid medium can be MMW medium with indoleacetic acid
(IAA), thidiazuron (TDZ) and silver nitrate (AgNO3). The solid medium can
further
comprise a selection agent against a gene encoded by the DNA construct. The
method can further comprise use of a chromosome doubling agent to produce a
doubled haploid transgenic plant. The doubling agent can be administered
within
one day after bombardment and can be administered for approximately 7 days.
Where the bombarded explant of the invention is a microspore-derived
embryo, the method may comprise: (a) culturing a microspore-derived embryo on
osmotic medium for a period of about half an hour to about 4 hours prior to
bombardment; (b) introducing a DNA construct by microprojectile bombardment
into an exposed surface of the microspore-derived embryo on osmotic medium;
(c)
continuing to culture the bombarded microspore-derived embryo on osmotic
medium for a period of about 4 hours to about 20 hours; (d) culturing said
bombarded microspore-derived embryo on regeneration media to produce a
regenerated embryo or tissue; and (e) regenerating a stably transformed
Brassica
plant from the regenerated embryo or tissue. The microspore-derived embryo can
be between about 11 and 20 days old. The microspore-derived embryo can be
between about 11 and 14 days old. The method can further comprise the step of
collecting the embryo using a pipette and transferring the embryo onto filter
paper
prior to step (a). Step (d) can comprise culturing the embryo on liquid medium
for a
first period of time and then on solid medium for a second period of time. The
first
period of time can be about 7 to 14 days. The method can further comprise an
optional step of excising a hypocotyl from the regenerated embryo and
cuituring the
hypocotyl on regeneration media. Additionally, the method can further comprise
a
step of selecting for a transformed embryo comprising culturing the embryo on
media supplemented with a selection agent against a gene encoded by the DNA
construct. The method can comprise use of a chromosome doubling agent to
produce a double haploid transgenic plant.
Where the bombarded explant of the invention is a poriion of a microspore-
derived embryo, the method may comprise: (a) culturing a hypocotyl excised
from a
microspore-derived embryo on osmotic medium for a period of about half an hour
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to about 4 hours prior to bombardment; (b) introducing a DNA construct by
microprojectile bombardment into an exposed surface of the microspore-derived
hypocotyl on osmotic medium; (c) continuing to culture the bombarded
microspore-
derived hypocotyl on osmotic medium for a period of about 4 hours to 20 hours;
(d)
cuituring said microspore-derived hypocotyl on a regeneration medium to
produce
regenerated embryos or tissues; and (e) regenerating a stably transformed
Brassica plant from said embryo or tissue. The microspore-derived hypocotyl
can
be excised from a microspore-derived embryo of between about 21 and 26 days.
The method can further comprise a step of culturing the microspore-derived
hypocotyl on a cell division induction medium comprising plant growth
regulators for
between about I to 20 hours prior to bombardment. Step (d) can comprise
culturing the embryo on a first solid medium for a first period of time and
then on a
second solid medium for a second period of time. The first solid medium can
comprise plant growth regulators for bud induction. The second solid medium
can
be free of plant growth regulators or comprises plant growth regulators for
shoot
formation. The method can further comprise a step of selecting a transformed
embryo or tissue comprising culturing the embryo or tissue on media
supplemented
with a selection agent against a gene encoded by the DNA construct. The method
can further comprise use of a chromosome doubling agent to produce a doubled
haploid transgenic plant.
49.Another aspect of the invention is to provide a Brassica cell or a stably
transformed Brassica plant produced by any one of the methods described above.
The plant or cell can be selected from Brassica napus, Brassica rapa, Brassica
juncea, Brassica oleracea, Brassica carinata and Brassica nigra. Progeny of
the
plant and cell are also provided.
DETAILED DESCRIPTION OF THE INVENTION
The invention is drawn to methods for transforming Brassica plants. The
methods find use in agriculture in the development of transgenic crop plants
with
improved agronomic characteristics. The methods find particular use in
introducing
desirable traits into a Brassica plant. Such new traits may be, for example,
resistance to an herbicide, resistance to pathogens and insects, modified seed
oil
composition and the like. The methods involve introducing a DNA construct into
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the genome of a cell of a Brassica plant and regenerating a stably transformed
plant from the cell.
A number of terms used herein are defined and clarified in the following
section.
By Brassica cell" is intended a cell from a Brassica plant or a cell that is
produced by in vitro culture methods and is descended from a cell from a
Brassica
plant.
By "somatic embryo" is intended an embryo that develops from a somatic
cell. The developmental process by which a somatic embryo develops from a cell
is known as "somatic embryogenesis." Such a "somatic embryo" is distinct from
a
"zygotic embryo" which develops from a zygote.
By "microspore-derived embryo" is intended an embryo that develops from a
microspore. Because it develops from a germ cell, such a m icrospo re-de rived
embryo" is distinct from both somatic and zygotic embryos which develop from
somatic cells and zygotes, respectively.
By "microspore-derived hypocotyl" is intended a hypocotyl of an embryo that
develops from a microspore.
By "adventitious" is intended an organ or other structure of a plant that does
not originate in the usual location on the plant body. For example, a shoot
that
originated from a hypocotyl of a microspore-derived embryo is an "adventitious
shoot."
By "canola" is intended a Brassica plant or oil from a Brassica plant wherein
the oil must contain less than 2% erucic acid and the solid component of the
seed
must contain less than 30 micromoles of any one or any mixture of 3-butenyl
glucosinolate, 4-pentenyl glucosinolate, 2-hydroxy-3 butenyl glucosinolate,
and 2-
hydroxy-4-pentenyl glucosinolate per gram of air-dry, oil free solid.
By "organogenesis" is intended the developmental process wherein a cell or
group of cells gives rise to an organ such as, for example, a shoot, a bud or
a root.
By "chromosome doubling" is intended that each of the chromosomes in a
cell is duplicated resulting in a doubling of the number of chromosomes in the
cell.
By "ploidy" is intended the number of complete sets of chromosomes in the
nucleus of a cell. A "haploid" cell has one set of chromosomes, and a
"diploid" cell
has two sets.
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By "effective amount" is intended an amount of an agent, compound or plant
growth regulator that is capable of causing the desired effect on an organism.
It is
recognized that an "effective amount" may vary depending on factors, such as,
for
example, the organism, the target tissue of the organism, the method of
administration, temperature, light, relative humidity and the like. Further,
it is
recognized that an "effective amount" of a particular agent may be determined
by
administering a range of amounts of the agent to an organism and then
determining
which amount or amounts cause the desired effect.
By "pre-incubated microspore-derived explant" is intended a microspore or
any tissue derived from the microspore (for example a microspore-derived
embryo
or a microspore-derived hypocotyl) that has been cultured for a period of time
of
between about 1 day and 30 days prior to bombardment. For example, a pre-
incubated microspore may be cultured for about two to ten days from the time
of
isolation of the microspore from a donor plant. For example, a pre-incubated
microspore-derived embryo may be cultured for a period of about 11 days to 20
days from the time of isolation of the microspore from a donor plant. For
example, a
pre-incubated microspore-derived hypocotyl may be cultured for a period of
about
21 to 26 days from the time of isolation of the microspore from a donor plant.
By "progeny" is intended descendents of a particular cell or plant which
comprise at least a portion of the transgene inserted at the locus of the
genome of
the TO plant cell. For example, progeny can be seeds developed on a plant and
plants derived from such seeds. For example, progeny of a plant include seeds
formed on TO, T1, T2 and subsequent generation plants, and plants derived from
such seeds. Progeny also includes seeds formed by cross pollination using
pollen
of a TO, T1, T2, T3, etc. plant. For example, the progeny can be the result of
selfing, outcrossing or backcrossing. The progeny can also include asexually
propagated plants or cells derived from TO, T1, T2, etc plants or cells that
include at
least a portion of the transgene inserted at the locus of the genome in the TO
plant
cell. For example, plants produced via cuttings, tissue culture, microspore
culture,
etc. that comprise at least a portion of the original transgene inserted at
the locus of
the genome of the TO plant cell are also considered progeny.
Methods are provided for transforming a Brassica plant. The methods
involve transforming a Brassica cell with a DNA construct by microprojectile
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bombardment. The methods further involve regenerating the transformed cell
into
a transformed Brassica plant. Such a transformed Brassica plant possesses at
least one copy of the DNA construct, or portion thereof, incorporated into its
genome. The transformed Brassica plants of the invention may be stably
transformed Brassica plants. Such transformed Brassica plants are capable of
producing at least one offspring that possesses at least one copy of the DNA
construct of the invention, or portion thereof, stably incorporated within its
genome.
Cells of the present invention may originate from (1) pre-incubated
microspores, (2) microspore-derived embryos or (3) microspore-derived
hypocotyls.
It is recognized that the cells of these tissues are most likely haploid. The
cells
may be diploid if the cells undergo spontaneous chromosome doubling, or if the
cells are subjected to chromosome doubling agents. Transformation of haploid
cells is advantageous because the resulting chromosome doubied transgenic
piant
is homozygous.
A DNA construct of interest is introduced into the ceil by microprojectile
bombardment. Microprojectile bombardment is also known by other terms,
including particle bombardment, microparticle bombardment, ballistic particle
acceleration and biolistic transformation. Generally, such methods involve
applying
to or coating the surface of microprojectiles with the DNA construct of
interest, and
then delivering the DNA-coated microprojectiles to the target tissue at a
velocity
sufficient to allow the particles to pass through cell walls and membranes and
thus,
enter plant cells. See, for example, Sanford et al., U.S. Patent No.
4,945,050;
Tomes et al., U.S. Patent No. 5,879,918; Tomes et al., U.S. Patent No.
5,886,244;
Bidney et al., U.S. Patent No. 5,932,782; Tomes et al. (1995) "Direct DNA
Transfer
into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell,
Tissue, and
Organ Culture: Fundamental. Methods, ed. Gamborg and Phillips (Springer-
Verlag,
Berlin); and McCabe et al. (1988) Biotechnology 6:923-926.
The methods of the invention do not depend on a particular DNA construct.
Any DNA construct that may be introduced into a cell by microprojectile
bombardment may be employed in the methods of the invention. DNA constructs
of the invention may comprise at least one nucleotide sequence of interest
operably
linked to a promoter that drives expression in a plant cell. DNA constructs
may
comprise a selectable marker gene and at least one additional nucleotide
sequence
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of interest operably linked to a promoter that drives expression in a plant
cell.
Additionally, DNA constructs may comprise a selectable marker gene and at
least
one additional nucleotide sequence that is capable of conferring a desired
trait on a
Brassica plant.
The methods of the present invention additionally comprise regenerating the
transformed cell of the invention into a stably transformed Brassica plant.
Regeneration of the transformed plant involves culturing the transformed cell
under
conditions that result in the growth and development of the transformed cell
into a
transformed plant. The transformed cell or descendents thereof may develop
into a
transformed embryo, particularly a transformed microspore-derived embryo or
somatic embryo which then develops into a transformed plant. Alternatively,
the
transformed cell and descendents thereof may develop into a transformed organ,
such as, for example, an adventitious shoot. It is recognized that
regenerating a
transformed Brassica plant from a transformed cell via an adventitious shoot
may
additionally involve the formation of callus before adventitious shoot
formation.
Such an adventitious shoot may be used to produce the stably transformed
Brassica plant by methods known in the art. Such methods generally invoive
culturing an adventitious shoot in a medium and environment which favors the
formation of adventitious roots on the adventitious shoot.
Methods for rooting adventitious shoots are known in the art. The methods
of the present invention do not depend on a particular method for rooting
transformed Brassica shoots. Any method known in the art for rooting
adventitious
shoots may be employed in the methods of the present invention. Generally,
rooting adventitious shoots will involve incubating shoots, for a period of
time, on a
medium that contains an effective amount of an auxin, such as, for example,
indolebutyric acid, to induce root formation. See, for example, Moloney et al.
(1989) Plant Cell Reports 8:238-242 and Radke et al. (1992) Plant Cell Reports
11:499-505. Rooted shoots may then be removed from culture, transferred to
soil
or potting medium and subjected to environmental conditions that favour
growth,
maturation and seed production.
It is recognized that the transformed embryos, transformed adventitious
organs, and transformed plants of the invention may be chimeric. That is, such
transformed embryos, organs and plants may be comprised of both transformed
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and non-transformed cells, or may be comprised of two or more differentially
transformed cells. It is further recognized that such chimeric plants may give
rise to
progeny plants that comprise a DNA construct of interest, or portion thereof,
stably
incorporated into the genomes of all of their somatic and germ line cells.
The methods of the invention involve the transformation of cells from
Brassica plants. The cells may be haploid cells. While haploid cells generally
do
not give rise to diploid plants, it is recognized that occasionally a haploid
cell may
spontaneously give rise to a diploid cell that is capable of developing into a
fertile
plant. If necessary, chromosome-doubling agents may be employed in the methods
of the invention to increase the ploidy of a haploid cell two fold. That is, a
haploid
cell becomes a diploid cell. Such a diploid cell may give rise to a fertile,
stably
transformed Brassica plant. The methods of the present invention do not depend
on a particular genetic mechanism of chromosome doubling. It is likely,
however,
that chromosome doubling results from chromosome duplication as would occur
for
example, during mitosis, but in the absence of cytokinesis.
Induced chromosome doubling of the invention involves administering an
effective amount of a chromosome-doubling agent to a cell, preferably a
haploid
cell. Any agent that is known to increase the ploidy of cells may be employed
in the
methods of the invention. Chromosome-doubling agents include, but are not
limited to, trifluralin, colchicine, oryzalin, amiprophosmethyl and pronamide.
Depending on the desired outcome, a chromosome-doubling agent may be
administered to a tissue, or a cell thereof, before, after, or both before and
after,
introducing a DNA construct into a cell by microprojectile bombardment. In
certain
methods of the invention, an effective amount of a chromosome-doubling agent
is
administered after bombardment.
The plants regenerated from transformed Brassica cells are referred to as
the TO generation or TO plants. The seeds produced by various sexual crosses
of
TO generation plants are referred to as TI progeny or T1 generations. When Tl
seeds are germinated, the resulting plants are also referred to as T1
generation.
Seeds produced on the TI plant or from crosses using T1 pollen, are referred
to as
T2 seeds, which give rise to T2 plants. Seeds produced on the T2 plant or from
crosses using T2 pollen, are referred to as T3 seeds. T3 seeds give rise to T3
plants. Accordingly, the generations progress through T4, T5, T6, etc. The
seeds
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and plants of the TI, T2, T3, T4, etc. can be analyzed to ensure successful
transmission of the transgene. Various sexual crosses are possible. For
example,
the plants can be selfed, outcrossed or backcrossed. Alternatively, the
transgenic
plants (TO, T1, T2, etc) can be propagated asexually, for example by cloning,
tissue
culture, cuttings, microspore culture, etc. as is known to those skilled in
the art.
In a first embodiment of the invention, methods are provided for transforming
Brassica pre-incubated microspores and regenerating stably transformed plants
therefrom.
The methods of the first embodiment involve bombarding pre-incubated
microspores with microprojectiles coated with a DNA construct of interest.
Microspores are isolated by methods that are known to those skilled in the
art. For
example, see Fukuoka et a/. (1996) Plant Physiol. 111:39-47; Keller et al.
(1987)
Proc. 7th Int. Rapeseed Congr. (Plant Breeding and Acclimatization Institute,
Poznan, Poland) pp. 152-157, Swanson et al. (1987) Plant Cell Reports 6: 94-97
and Baillie et al. (1992) Plant Cell Reports 11: 234-237. The microspores are
haploid. The microspores may be isolated and cultured in a medium with a high
level of sucrose, for example 17% sucrose, for 2 to 3 days. The high level of
sucrose is recommended to ensure the integrity of the microspores immediately
after isolation. Further, high osmotic stress would have a positive effect on
embryogenesis induction (Maraschin et al., 2005 J Exp Bot 56: 1711-1726 and
Prem et al. 2005 In Vitro Cell. Dev. Biol. - Plant 41:266-273). The
microspores are
then cultured for 4 to 8 days in medium containing a reduced level of sucrose,
for
example in the range of 10% sucrose to promote microspore division.
Accordingly,
a pre-incubation period of 2 to 10 days is within the scope of the invention.
After the pre-incubation period, the pre-incubated microspores are collected
in a manner to enrich for viable and embryogenic microspores. This can be
done,
for example, by using a NitexTM sieve of 15 to 48 pm in pore size. The pore
size
may be between 15 and 25 {am. The embryogenic microspores can also be
enriched by Percoll gradient centrifugation (Touraev 1996 Sex Plant Reprod 9:
209-215). Percoll concentration is between 35 to 45%. The NitexTM sieve
holding
the pre-incubated microspores is then placed on an osmotic treatment medium
prior to bombardment, during bombardment and for a period of time after
bombardment. The osmotic treatment induces slight plasmolysis of the
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microspores to ensure they will not burst due to the bombardment procedure.
During bombardment, a surface of the microspores may be exposed to the path of
the bombarding particles coated with DNA to facilitate entry of the particles
and
also to prevent any sudden influx of medium (i.e. the surface of the
microspores
that is exposed is not embedded in the medium). The osmotic treatment
facilitates
this. For example, the pre-incubated microspores may be subjected to the
osmotic
treatment for 1 to 2 hours prior to bombardment, during the bombardment, and
for
1 to 24 hours after bombardment. For example, the osmotic treatment may
comprise placing the pre-incubated microspores on medium containing between
0.8 to 1.6% PhytagelTM or agar, between 17 and 19% sucrose and lg/l MES (2-[N-
Morpholino] ethanesulfonic acid). The osmotic treatment can also be done by
placing microspores (optionally on sieves) in a petri dish (3.5 cm in
diameter) prior
to bombardment, during the bombardment and after the bombardment. A piece of
filter paper (3.2 cm in diameter) wet with NLN-13S medium is placed in the
petri
dish to prevent microspore dehydration.
Foilowing bombardment, the microspores and sieve can be cultured in
medium containing a doubling agent and a high level of sucrose, for example
13%
sucrose, for about 7 days.
If the DNA construct of interest comprises a selectable marker gene, the
bombarded pre-incubated microspores may be transferred to medium containing
an appropriate selective agent for that particular selectable marker gene.
Such a
transfer may occur immediately after bombardment or after a period of time.
For
example, the transfer may occur between 0 and about 30 days after bombardment.
During the selection stage, the pre-incubated microspores may be sub-
cultured in selection NLN medium, which may contain a reduced sucrose content,
for example 6.5% and growth regulators, for example cytokinins and auxins.
Selection may be conducted in the light. The pre-incubated microspores may
then
be monitored for the appearance of transformed embryos and/or adventitious
shoots. Such transformed embryos and/or adventitious shoots may then be
cultured in shoot regeneration medium that may contain MS or B5 components and
further, may also contain selection agents (for example, kanamycin or
glyphosate)
with or without plant growth regulators. The regenerated shoots are rooted in
B5
medium containing 0.1 mg/I GA3.
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In a second embodiment of the invention, methods are provided for
transforming cells from microspore-derived embryos with microprojectiles
coated
with a DNA construct of interest. Methods are known in the art for producing
embryos from Brassica microspores. See Fukuoka et al. (1996) Plant Physiol.
111:39-47 and Keller et al. (1987) Proc. 7th lnt. Rapeseed Congr. (Plant
Breeding
and Acclimatization Institute, Poznan, Poland) pp. 152-157. Like the
microspores
themselves, the cells comprising such microspore-derived embryos are haploid.
In
the methods of the invention, whole microspore-derived embryos are bombarded
with DNA-coated microprojectiles. The microspore-derived embryos may be
greater than 10 days old and approximately greater than 1.5 mm in size. The
microspore-derived embryos can be between 11 and 20 days old. The embryos
may be globular or heart shaped. The embryos are placed on osmotic medium
prior to, during and after bombardment for the same reasons as discussed
above.
At least one surface of the embryos should be exposed to the path of the
bombarded particles (i.e. not in the medium) during bombardment to facilitate
entry
of the particle and to avoid any sudden influx of medium into the cell. For
example,
the embryos may be subjected to the osmotic treatment for approximately 4
hours
prior to bombardment and for approximately 20 hours (for example, overnight)
after
bombardment. The osmotic treatment may comprise, for example, a medium
containing 17 to 19% sucrose and 1.5% agar, and acts to prevent the cells of
the
embryos from bursting during and after bombardment. Alternatively, the osmotic
treatment may comprise placing the embryos on a petri dish having a wet filter
paper. The embryos are then transferred to regeneration medium. The
regeneration media may inciude, but are not limited to, B5 media, MS-based
media
(MS salts with organics, 2% (w/v) sucrose, 0.6% (w/v) Sigma agar, pH 5.8).
Embryo-derived hypocotyls may be excised and cultured in selection medium to
induce transgenic shoots. Typically, a microspore-derived embryo gives rise to
a
single or a few adventitious shoots as a result of growth from the apical
meristem or
hypocotyl area. Methods of the second and third embodiments can involve
adventitious shoot regeneration of the microspore-derived embryos and
microspore-derived hypocotyls. Such methods find use in increasing the number
of
transformed plants recovered from a transformation attempt.
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Adventitious shoot regeneration involves the formation of multiple shoots
arising from a microspore-derived embryo. Thus, a single microspore-derived
embryo can yield multiple transformed shoots from a transformation. Typically,
all
of the transformed shoots that arise from a single microspore-derived embryo
are
thought to be an independent transformant. That is, each transformed shoot is
derived from an independently transformed cell and thus, is genetically
distinct. For
the purposes of this investigation, however, all multiple events from each
embryo
were combined.
Methods of adventitious shoot regeneration are known in the art. While the
methods of the present invention do not depend on a particular method of
adventitious shoot regeneration, the methods may involve subjecting the
microspore-derived embryos to an effective amount of cytokinin to induce
adventitious shoots. Adventitious shoot regeneration may be accomplished
within
less than about 30 days after administering a cytokinin to the microspore-
derived
embryos. Adventitious shoot regeneration may be accomplished within less than
about 10 days after administering the cytokinin. The methods of secondary
regeneration of the present invention may additionally involve subjecting the
microspore-derived embryos to an effective amount of an auxin. In exemplary
methods, an effective amount of a cytokinin is administered, with or without
an
effective amount of an auxin, to the microspore-derived embryos following
bombardment to induce adventitious shoot regeneration.
If the DNA construct utilized in methods of the second embodiment
comprises a selectable marker gene, selection may be applied immediately after
bombardment or after a period of time of less than 2 day to about 30 days.
Selection may be applied by subjecting the microspore-derived embryos to an
effective amount of an appropriate selective agent for the selectable marker
gene
of the DNA construct of interest. An effective amount of the selective agent
may be
added to the medium on which the microspore-derived embryo is cultured. The
selective agent may be administered alone or in combination with one or more
other compounds such as a chromosome-doubling agent or a plant growth
regulator.
In a third embodiment of the invention, methods are provided for particle
bombardment of microspore-derived hypocotyls. As discussed above, microspores
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are isolated and cultured as is known to those skilled in the art.
Approximately 21
days after culture, when the embryos are generally torpedo shaped, the culture
medium is diluted with fresh culture medium and the microspores are allowed to
culture for approximately 5 more days. The majority of the embryos are
generally
at the cotyledon stage after 26 days in culture. Hypocotyl sections from the
embryos are excised and cultured for a period of time on medium supplemented
with plant growth regulators to induce cell division. For example, the excised
hypocotyls may be cultured overnight on MMW + 4 mg/I BAP + 0.25 mg/I NAA.
Prior to bombardment, during bombardment and after bombardment, the
excised hypocotyls are subjected to osmotic treatment as described above. In
addition, a surface of the hypocotyls in direct line with the bombardment
route is
exposed to the path of the bombarding particle (i.e. the surface of the
hypocotyls
that is in the direct line with the bombardment route is not embedded in the
medium) to facilitate entry of the particle coated with DNA and to avoid any
sudden
influx of medium. The osmotic medium may comprise 17 to 19% sucrose, 1.5%
agar and 1g/I MES. The hypocotyls may be cultured on the osmotic medium for 4
hours prior to bombardment and overnight after bombardment. After osmotic
treatment, the hypocotyls may be transferred to bud induction medium (for
example, MMW + 4 mg/I BAP + 25-100 mg/I KAN). A bud can be an immature
shoot, leaf, embryo or flower. Hypocotyls comprising adventitious buds may
then
be transferred to shoot regeneration medium (for example, MMW without
hormones, or MMW + 0.2 mg/I BAP). A selection agent may be added to any of the
media after bombardment.
Additionally, the methods of the first, second, and third embodiments may
comprise administering an effective amount of a chromosome doubling agent to
the
culture medium before, or optionally after, bombardment. Adding a chromosome
doubling agent is not necessary in all cases, because the rate of spontaneous
doubling can be high, especially in the embodiments employing microspore-
derived
embryos and microspore-derived hypocotyls. Such chromosome-doubling agents
and methods of use are known to those skilled in the art and were discussed
above.
The methods of the present invention involve the use of plant growth
regulators such as, for example, auxins and cytokinins. The plant growth
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WO 2007/055687 PCT/US2005/040646
regulators of the invention include, but are not limited to, both free and
conjugated
forms of naturally occurring plant growth regulators. Additionally, the plant
growth
regulators of the invention encompass synthetic analogues and precursors of
such
naturally occurring plant growth regulators.
Naturally occurring and synthetic analogues of auxins include, but are not
limited to, indoleacetic acid (IAA), 3-indolebutyric acid (IBA), a-
napthaleneacetic
acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), 4-(2,4-dichlorophenoxy)
butyric
acid, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), (4-chloro-2-methylphenoxy)
acetic
acid (MCPA), 4-(4-chloro-2-methylphenoxy) butanoic acid (MCPB), mecoprop,
dicloprop, quinclorac, picloram, triclopyr, clopyralid, fluroxypyr and
dicamba.
Naturally occurring and synthetic analogues of cytokinins include, but are not
limited to, kinetin, thidiazuron (TDZ), zeatin, zeatin riboside, zeatin
riboside
phosphate, dihydrozeatin, isopentyl adenine and 6-benzyladenine (BAP).
The methods of the present invention may include use of G418 disulfate salt
(GibcoTM), also sold as GeneticinTM from Fluka as a selection agent or
glyphosate
as a selection agent. After bombardment of pre-incubated microspores,
selection
may be done in liquid medium in dark first and then under low light intensity
(for
example, approximately 240 foot-candles or 2,583 lux). However, other
selection
agents, as is known to those skilled in the art, can be used. For example,
kanamycin (Beck et al. (1982) Gene 19:327-336; Mazodier et al. (1985) Nucleic
Acids Res. 13:195-205), and other herbicides, like BastaTM and ChlorsulfuronT
".
Stable transgenic plants may be confirmed by polymerase chain reaction
(PCR) analysis and Southern blot hybridization analysis.
The use of the term "DNA constructs" herein is not intended to limit the
present invention to nucleotide constructs comprising DNA. Those of ordinary
skill
in the art will recognize that nucleotide constructs, particularly
polynucleotides and
oligonucleotides, comprised of ribonucleotides and combinations of
ribonucleotides
and deoxyribonucleotides may also be employed in the methods disclosed herein.
Thus, the DNA constructs of the present invention encompass all nucleotide
constructs which can be employed in the methods of the present invention for
transforming Brassica plants including, but not limited to, those comprised of
deoxyribonucleotides, ribonucleotides and combinations thereof. Such
deoxyribonucleotides and ribonucleotides include both naturally occurring
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WO 2007/055687 PCT/US2005/040646
molecules and synthetic analogues. The DNA constructs of the invention also
encompass all forms of nucleotide constructs including, but not limited to,
single-
stranded forms, double-stranded forms, hairpins, stem-and-loop structures and
the
like.
Furthermore, it is recognized that the methods of the invention may employ
a DNA construct that is capable of directing, in a transformed plant, the
expression
of at least one protein, or at least one RNA, such as, for example, an rRNA, a
tRNA
or an antisense RNA that is complementary to at least a portion of an mRNA.
Typically such a DNA construct is comprised of a coding sequence for a protein
or
an RNA operably linked to 5' and 3' transcriptional regulatory regions.
Alternatively, it is also recognized that the methods of the invention may
employ a
DNA construct 'that is not capable of directing, in a transformed plant, the
expression of a protein or RNA.
In addition, it is recognized that methods of the present invention do not
depend on the incorporation of the entire DNA construct into the genome, only
that
the genome of the Brassica plant is altered as a result of the introduction of
the
DNA construct into a Brassica cell. For example, alterations to the genome
include
additions, deletions and substitution of nucleotides in the genome. While the
methods of the present invention do not depend on additions, deletions, or
substitutions of any particular number of nucleotides, it is recognized that
such
additions, deletions or substitutions comprise at least one nucleotide.
The DNA constructs of the invention also encompass nucleotide constructs,
that may be employed in methods for altering or mutating a genomic nucleotide
sequence in an organism, including, but not limited to, chimeric vectors,
chimeric
mutational vectors, chimeric repair vectors, mixed-duplex oligonucleotides,
self-
complementary chimeric oligonucleotides and recombinogenic oligonucleobases.
Such nucleotide constructs and methods of use, such as, for example,
chimeraplasty, are known in the art. Chimeraplasty involves the use of such
nucleotide constructs to introduce site-specific changes into the sequence of
genomic DNA within an organism. See, U.S. Patent Nos. 5,565,350; 5,731,181;
5,756,325; 5,760,012; 5,795,972; and 5,871,984. See also, WO 98/49350, WO
99/07865, WO 99/25821 and Beetham et al. (1999) Proc. Nafl. Acad. Sci. USA
96:8774-8778.
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Additionally, the term "DNA-coated microprojectiles" used herein is not
intended to limit the methods of the present invention to microprojectiles
coated
with DNA. Rather, the term "DNA-coated microprojectiles" as used herein
encompasses microprojectiles coated with any one or more of the DNA constructs
of the invention as described supra.
The DNA constructs of the invention may be comprised of expression
cassettes for expression in the Brassica plant of interest. The expression
cassette
may include 5' and 3' regulatory sequences operably linked to a gene of
interest.
By "operably linked" is intended a functional linkage between a regulatory
sequence and a second sequence, wherein the regulatory sequence affects
initiation and mediation of transcription of the DNA sequence corresponding to
the
second sequence. Generally, operably linked means that the nucleic acid
sequences being linked are contiguous and, where necessary to join two protein
coding regions, contiguous and in the same reading frame. The cassette may
additionally contain at least one additional gene to be cotransformed into the
organism. Alternatively, the additional gene(s) can be provided on multiple
expression cassettes.
Such an expression cassette is provided with a plurality of restriction sites
for insertion of the gene of interest sequence to be under the transcriptional
regulation of the regulatory regions. The expression cassette may additionally
contain selectable marker genes.
The expression cassette may include in the 5'-3' direction of transcription, a
transcriptional and translational initiation region, a gene of interest and a
transcriptional and translational termination region functional in plants. The
transcriptional initiation region, the promoter, may be native (or analogous)
or
foreign (or heterologous) to the plant host. Additionally, the promoter may be
the
natural sequence or alternatively a synthetic sequence. By "foreign" is
intended
that the transcriptional initiation region is not found in the native plant
into which the
transcriptional initiation region is introduced. As used herein, a chimeric
gene
comprises a coding sequence operably linked to a transcription initiation
region that
is heterologous to the coding sequence.
While it may be preferable to express the gene of interest using
heterologous promoters, the native promoter sequences may be used. Such
CA 02629284 2008-05-09
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constructs would change expression levels of the gene of the interest in the
plant or
plant cell. Thus, the phenotype of the plant or plant cell is altered.
The termination region may be native with the transcriptional initiation
region, may be native with the operably linked DNA sequence of interest, or
may be
derived from another source. Convenient termination regions are available from
the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline
synthase termination regions. See also Guerineau et al. (1991) Mol. Gen.
Genet.
262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes
Dev.
5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990)
Gene
91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et
al.
(1987) Nucleic Acid Res. 15:9627-9639.
Where appropriate, the gene(s) may be optimized for increased expression
in the transformed plant. That is, the genes can be synthesized using plant-
preferred codons for improved expression. See, for example, Campbell and Gowri
(1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage.
Methods are available in the art for synthesizing plant-preferred genes. See,
for
example, U.S. Patent Nos. 5,380,831, and 5,436,391, and Murray et al. (1989)
Nucleic Acids Res. 17:477-498.
Additional sequence modifications are known to enhance gene expression in
a cellular host. These include elimination of sequences encoding spurious
polyadenylation signals, exon-intron splice site signals, transposon-like
repeats,
and other such well-characterized sequences that may be deleterious to gene
expression. The G-C content of the sequence may be adjusted to levels average
for a given cellular host, as calculated by reference to known genes expressed
in
the host cell. When possible, the sequence is modified to avoid predicted
hairpin
secondary mRNA structures. In addition, the gene can undergo gene shuffling to
enhance expression. For example, the glyphosate acetyl transferase gene used
in
the examples underwent gene shuffling (Castle et al. (2004) Science 304:1151-
1154 and WO 02/36782A2).
The expression cassettes may additionally contain 5'-leader sequences in the
expression cassette construct. Such leader sequences can act to enhance
translation. Translation leaders are known in the art and include:
picornavirus
leaders, for example, EMCV leader (Encephalomyocarditis 5'-noncoding region)
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(Elroy-Stein et al. (1989) PNAS USA 86:6126-6130); potyvirus leaders, for
example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986)
Virology 154: 9-20); MDMV leader (Maize Dwarf Mosaic Virus); Virology 154:9-
20),
and human immunoglobulin heavy-chain binding protein (BiP), (Macejak et al.
(1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of
alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625);
tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology
of
RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus
leader (MCMV) (Lommel et al. (1991) Virology 81:382-385). See also, Della-
Cioppa et al. (1987) Plant Physiol. 84:965-968. Other methods known to enhance
translation can also be utilized, for example, introns, and the like.
In preparing the expression cassette, the various DNA fragments may be
manipulated, so as to provide for the DNA sequences in the proper orientation
and,
as appropriate, in the proper reading frame. Toward this end, adapters or
linkers
may be employed to join the DNA fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of superfluous
DNA,
removal of restriction sites, or the like. For this purpose, in vitro
mutagenesis,
primer repair, restriction, annealing, resubstitutions, e.g., transitions and
transversions, may be involved.
A number of promoters can be used in the practice of the invention. The
promoters can be selected based on the desired outcome. The nucleic acids can
be combined with constitutive, tissue-preferred, or other promoters for
expression
in Brassica plants.
Such constitutive promoters include, for example, the core promoter of the
Rsyn7 (U.S. Patent No. 6,072,050); the core CaMV 35S promoter (Odell et al.
(1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant CeII 2:163-
171);
ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619- 25 632 and
Christensen
et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor.
Appl.
Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS
promoter (U.S. Patent No. 5,659,026), SCP (WO 97/47756A1, WO 99/438380);
H2b (Rasco-Gaunt et al. (2003) Plant Cell Rep. 21:569-576); SCP1 (US Patent
No.
6,677,503 131) and the like. Other constitutive promoters include, for
example, U.S.
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Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;
5,268,463; and 5,608,142.
Tissue-preferred promoters can be utilized to target enhanced expression of
the gene of interest within a particular plant tissue. Tissue-preferred
promoters
include Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997)
Plant Cell Physiol. 38(7):792-803; Hanson et al. (1997) Mol. Gen Genet.
254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart
et al.
(1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol.
112(2):525-535; Canevascini et al. (1996) Plant Physiol 112(2):513-524;
Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results
Probi. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mot Biol.
23(6):1129-1138;
Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-
Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters can be modified, if
necessary, for weak expression.
"Seed-preferred" promoters include both "seed development" promoters
(those promoters preferentially active during seed development such as
promoters
of seed storage proteins) as well as "seed-germinating" promoters (those
promoters preferentially active during seed germination). See Thompson et al.
(1989) BioEssays 10:108. Such seed-preferred promoters include, but are not
limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein);
milps
(myo-inositol-l-phosphate synthase); and celA (cellulose synthase) (see U.S.
Patent No. 6,225,529). For dicots, seed-specific promoters include, but are
not
limited to, bean P-phaseolin (Chandrasekharan et al., 2003 Plant J. 33: 853-
866),
napin, P-conglycinin (Chamberland et al. 1992 Plant Mol. Biol. 19: 937-949),
soybean lectin, cruciferin, and the like.
Various changes in phenotype are of interest including modifying the fatty
acid composition in a plant, altering the amino acid content of a plant,
altering a
plant's pathogen defense mechanism, enhancing tolerance to abiotic stress, and
the like. These results can be achieved by providing expression of
heterologous
products or increased expression of endogenous products in plants.
Alternatively,
the results can be achieved by providing for a reduction of expression of one
or
more endogenous products, particularly enzymes or cofactors in the plant.
These
changes result in a change in phenotype of the transformed plant.
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Genes or nucleotide sequences of interest are reflective of the commercial
markets and interests of those involved in the development of the crop. Crops
and
markets of interest change, and as developing nations open up world markets,
new
crops and technologies will emerge also. In addition, as our understanding of
agronomic traits and characteristics such as yield and heterosis increase, the
choice of genes for transformation will change accordingly. General categories
of
genes of interest include, for example, those genes involved in information,
such as
zinc fingers, those involved in communication, such as kinases, and those
involved
in housekeeping, such as heat shock proteins. More specific categories of
transgenes, for example, include genes encoding important traits for
agronomics,
insect resistance, disease resistance, herbicide resistance, sterility, grain
characteristics, and commercial products. Genes of interest include,
generally,
those involved in oil, starch, carbohydrate, or nutrient metabolism as well as
those
affecting kernel size, sucrose loading, and the like.
Agronomically important traits such as oil, starch, and protein content can be
genetically altered by methods of the invention in addition to using
traditional
breeding methods. Modifications include increasing content of oleic acid,
saturated
and unsaturated oils, increasing levels of lysine and sulfur, providing
essential
amino acids, and also modification of starch. Hordothionin protein
modifications
are described in U.S. Patent Nos. 5,990,389, 5,885,801, 5,885,802, and
5,703,409.
Another example is lysine and/or sulfur rich seed protein encoded by the
soybean
2S albumin described in U.S. Patent No. 5,850,801, and the chymotrypsin
inhibitor
from barley, described in Williamson et al. (1987) Bur. J. Biochem. 165:99-106
Derivatives of the coding sequences can be made by site-directed
mutagenesis to increase the level of preselected amino acids in the encoded
polypeptide. For example, the gene encoding the barley high lysine polypeptide
(BHL) is derived from barley chymotrypsin inhibitor, WO 98/20133. Other
proteins
include methionine-rich plant proteins such as from sunflower seed (Lilley et
al.
(1989) Proceedings of the World Congress on Vegetable Protein Utilization in
Human Foods and Animal Feedstuffs, ed. Applewhite (American Oil Chemists
Society, Champaign, Illinois), pp. 497-502); corn (Pedersen et al. (1986) J.
Biol.
Chem. 261:6279; Kirihara et al. (1988) Gene 71:359); and rice (Musumura et al.
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(1989) Plant Mol. Biol. 12:123). Other agronomically important genes encode
latex,
Floury 2, growth factors, seed storage factors, and transcription factors.
Insect resistance genes may encode resistance to pests that have great
yield drag such as rootworm, cutworm, European corn borer, and the like. Such
genes include, for example, Bacillus thuringiensis toxic protein genes (U.S.
Patent
Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; and Geiser et al.
(1986) Gene 48:109); lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825;
Ahman et al. 2000 W00001 223); and the like.
Genes encoding disease resistance traits include detoxification genes, such
as against fumonosin (U.S. Patent No. 5,792,931); avirulence (avr) and disease
resistance (R) genes (Jones et a/. (1994) Science 266:789; Martin et al.
(1993)
Science 262:1432; and Mindrinos et al. (1994) Cell 78:1089); and the like.
Genes
conferring resistance to Sclerotinia have been introduced into sunflower and
Brassica (U.S. Patent No. 6,441,275 B1). An endochitinase gene under a
constitutive promoter was introduced into canola (Brassica napus var.
oleifera)
inbred line. Progeny from the transformed plants were challenged using three
different fungal pathogens (Cylindrosporium concentricum, Phoma lingam,
Sclerotinia sclerotiorum) in field trials. The plants exhibited an increased
tolerance
to disease as compared with the nontransgenic parental plants (Grison et al.
(1996)
Nature Biotechnology 14: 643-646). Additional disease resistance genes are
discussed in Stewart and Broadway, 2005 (US6927322); Salmeron et al. 2003
(US6528702) and Chye and Zhao 2002 (US20030097682).
Herbicide resistance traits may include genes coding for resistance to
herbicides that act to inhibit the action of acetolactate synthase (ALS), in
particular
the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene
containing mutations leading to such resistance, in particular the S4 and/or
Hra
mutations), genes coding for resistance to herbicides that act to inhibit
action of
glutamine synthase, such as phosphinothricin or Basta (e.g., the bar gene),
or
other such genes known in the art. The ALS-gene mutants encode resistance to
the herbicide chlorsulfuron (Swanson et al (1989) Theor Appl Genet 78:525-530,
EP0257993 BI). The glyphosate acetyl transferase (GAT) gene confers resistance
to glyphosate (Castle et al. (2004) Science 304:1151-1154).
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Sterility genes can also be encoded in an expression cassette and provide
an alternative to physical emasculation. Examples of genes used in such ways
include male tissue-preferred genes and genes with male sterility phenotypes
such
as avidin and streptavidin, described in U.S. Patent No. 5,962, 769 (Aibertsen
et
al., 1999) and Barnase (Block and Debrouwer (1993) Planta 189: 218-225) Other
genes include kinases and those encoding compounds toxic to either male or
female gametophytic development.
The quality of seed is reflected in traits such as levels and types of oils,
saturated and unsaturated, quality and quantity of essential amino acids, and
levels
of cellulose. For example, U.S. Patent Nos. 5,990,389, 5,885,801, 5,885,802,
and
5,703,409, provide descriptions of modifications of proteins for desired
purposes.
Commercial traits can also be encoded on a gene or genes that could
increase for example, starch for ethanol production, or provide expression of
proteins. Another important commercial use of transformed plants is the
production
of polymers and bioplastics such as described in U.S. Patent No. 5,602,321.
Genes such as R-Ketothiolase, PHBase (polyhydroxybutryrate synthase), and
acetoacetyl-CoA reductase (see Schubert et al. (1988) J. Bacleriol. 170:5837-
5847)
facilitate expression of polyhydroxyalkanoates (PHAs).
Exogenous products include plant enzymes and products as well as those
from other sources including prokaryotes and other eukaryotes. Such products
include enzymes, cofactors, hormones, and the like. The level of proteins,
particularly modified proteins having improved amino acid distribution to
improve
the nutrient value of the plant, can be increased. This is achieved by the
expression of such proteins having enhanced amino acid content.
It is recognized that a DNA construct of the present invention may comprise
an antisense construction complementary to at least a portion of a messenger
RNA
(mRNA) of a gene of interest. Antisense nucleotides are constructed to
hybridize
with the corresponding mRNA. Modifications of the antisense sequences may be
made as long as the sequences hybridize to and interfere with expression of
the
corresponding mRNA. in this manner, antisense constructions having 70%, 80%,
or 85% or more sequence identity to the complementary sequences may be used.
Furthermore, portions of the antisense nucleotides may be used to disrupt the
expression of the target gene. Generally, sequences of at least 50
nucleotides,
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WO 2007/055687 PCT/US2005/040646
100 nucleotides, 200 nucleotides, or greater may be used. Typically, such
antisense constructions will be operably linked to a promoter that drives
expression
in a plant.
The DNA constructs of the invention may also be employed in sense
suppression methods to suppress the expression of endogenous genes in plants.
Methods for suppressing gene expression in plants using nucleotide sequences
in
the sense orientation are known in the art. The methods generally involve
transforming plants with a DNA construct comprising a promoter that drives
expression in a plant operably linked to at least a portion of a nucleotide
sequence
that corresponds to the transcript of the endogenous gene. Typically, such a
nucleotide sequence has substantial sequence identity to the sequence of the
transcript of the endogenous gene, for exampie, greater than about 65%, 85% or
95% sequence identity. See, U.S. Patent Nos. 5,283,184 and 5,034,323.
Generally, the expression cassette will comprise a selectable marker gene
for the selection of transformed cells. Selectable marker genes are utilized
for the
selection of transformed cells or tissues. Marker genes include genes encoding
antibiotic resistance, such as those encoding neomycin phosphotransferase II
(NPTII), and hygromycin phosphotransferase (HPT). Genes conferring resistance
to herbicidal compounds may also be used, such as glyphosate acetyl
transferase,
glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-
dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992) Curr. Opin.
Biotech. 3:506-511; Christopherson et al. (1992) Proc. Nati. Acad. Sci. USA
89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol.
Microbiol.
6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.
(1987)
Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell
52:713-722; Deuschle et al. (1989) Proc. Natl. Acad. Sci. USA 86:5400-5404;
Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al.
(1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of
Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921;
Labow
et al. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc.
Natl. Acad.
Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-
5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-
Wissman
(1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb et al. (1991)
Antimicrob.
27
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WO 2007/055687 PCT/US2005/040646
Agents Chemother. 35:1591-1595; Kleinschnidt et al. (1988) Biochemistry
27:1094-
1104; Bonin (1993) Ph.D. Thesis University of Heidelberg; Gossen et al. (1992)
Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob.
Agents
Chemother. 36:913-919; Hlavka et al. (1985) Handbook of Experimental
Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature
334:721-
724.
In addition, the expression cassette may comprise a screenable marker
gene, for example the gene encoding P-glucuronidase (GUS) (Jefferson et al.
(1986) Proc. Natl. Acad. Sci. USA 83:8447-8451; Jefferson et al. (1987) EMBO
J.
6:3901-3907) or the gene encoding the green fluorescent protein (GFP) (Chalfie
et
al. (1994) Science 263: 802-805).
The above list of selectable marker genes and screenable marker genes is
not meant to be limiting. Any selectable and/or screenable marker gene can be
used in the present invention.
Brassica plants of the invention include, but are not limited to, Brassica
carnata (Ethiopian mustard), Brassica juncea (leaf mustard), Brassica napus
(rape), Brassica napus var. rapifera (Swedish turnip), Brassica nigra (black
mustard), Brassica oleracea, Brassica oleracea var. acephala (kale), Brassica
oleracea var. alboglabra (Chinese kale), Brassica oleracea var, hotrytis
(cauliflower, heading broccoli), Brassica oleracea var. capitata (cabbage),
Brassica
oleracea var. gemmifera (Brussel sprouts), Brassica oleracea var. gongylodes
(Kohlrabi), Brassica rapa (field mustard; also known as Brassica campestris),
Brassica rapa subsp. chinensis (bok-choy), and Brassica rapa subsp. pekinensis
(Chinese cabbage).
In certain embodiments of the invention, the Brassica plants of the invention
are oilseed Brassica plants. Such oilseed Brassica plants are used for oil
production and include but are not limited to, Brassica juncea, Brassica napus
and
Brassica rapa. The Brassica plants may be canola plants. Such canola plants
are
selections of oilseed Brassica plants (Brassica rapa, Brassica napus and
Brassica
juncea) that contain low levels of both erucic acid and glucosinolates in
their seeds.
Canola oil must contain less than 2% erucic acid and the solid component of
the
seed must contain less than 30 micromoles of any one or any mixture of 3-
butenyl
glucosinolate, 4-pentenyl glucosinolate, 2-hydroxy-3 butenyl glucosinolate,
and 2-
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hydroxy-4-pentenyl glucosinolate per gram of air-dry, oil free solid. The
seeds of
such canola plants are favored for the extraction of edible oils.
EXPERIMENTAL
Microspore-derived embryos
Experiments I to 2 describe the work done using microspore-derived
embryos as the target tissue. Microspores were isolated as is known to those
skilled in the art, for example, see Swanson et al. 1987 and cultured in NLN
medium (see Section entitled "Media Recipes" for components of all media used
in
this invention) for approximately 11 to 14 days. However, the microspore-
derived
embryos can be cultured for between 11 to 20 days. The embryos were produced
from the microspores and were detectable with the naked eye. The size of the
microspore-derived embryos was generally smaller than 1 mm and the embryos
were globular or heart shaped.
The microspore-derived embryos were collected to enrich for viable
embryos. For example, this may be done using a pipette and transferring the
embryos to a filter paper or membrane, for example Gelman T"' membrane (Prod.
No. 60110). The filter paper or membrane can have a 0.8pm pore size. The
embryos and membrane were cultured on osmotic medium for example, the
medium may contain 17 to 19% sucrose + 0.8 to 1% agar + I g/l MES, pH 6Ø The
embryos were subjected to the osmotic treatment before, during and after
bombardment. For exampie, the embryos were subjected to the osmotic treatment
for 4 hours prior to bombardment. The DNA construct used in bombardments was
PHP18644. PHP18644, and other vectors used are described in the Table 9. The
bombardment was done as is known to those skilled in the art, using
approximately
10 ng to 5pg of DNA per preparation, 15 pg to 300 pg of gold particles of
approximately 0.4-1.0 micron per shot, CaC12 at 0.5M to 2.5M and a rupture
disk of
650, 900 or 1100 psi. After bombardment, the embryos were cultured on the
osmotic medium for 4 hours to 20 hours (approximately overnight) and then
transferred to liquid NLN medium. The bombarded embryos were subsequently
cultured for 7 to 14 days. The bombarded embryos or hypocotyls excised from
the
embryos at age 3 to 4 weeks were transferred from liquid NLN medium to solid
selection medium MMW + IAA + TDZ + K25-50 (embryos) or MMW + BAP + K25-
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50 (hypocotyls) for bud induction. The resistant regenerated buds were excised
and cultured on MMW-H + K50-100 for plant regeneration.
Experiment 1: Effect of various rupture disks on GUS transient
expression using microspore-derived embryos
The objective was to screen different rupture disks to determine those that
would result in the highest transformation efficiency.
Part A: Fourteen day old microspore-derived embryos were pre-incubated
for 4 hours on osmotic medium containing 17% sucrose, 1 g/l MES and 10 g/I
agar
with pH 6Ø The microspore-derived embryos were bombarded with PHP18644
precipitated on goid particles (100 pg/shot). The bombarded embryos were
cultured for 4 hours after bombardment on the osmotic medium containing 17%
sucrose, 1 g/l MES and 10 g/l agar with pH 6.0 and then cultured in NLN
medium.
Rupture disks of 450 psi, 650 psi and 900 psi were tested.
Transient transformation efficiency was determined by analyzing the
bombarded embryos using the GUS assay, as is known to those skilled in the
art.
Table 1 shows the results of the GUS assay. Rupture disk 900 psi produced the
greatest number of cells expressing GUS in this experiment.
Table 1. Effect of cultivar and rupture disk on GUS transient expression using
microspore-derived embryos
Rep # Rupture disk # of bombarded GUS dots/embryos
embryos
Rep 1 450 psi 200 4
650 psi 193 5.7
900 psi 207 2.9
Rep 2 450 psi 107 7.5
650 psi 54 20.4
900 psi 51 31.4
Rep 3 450 psi 39 69.2
650 psi 39 76.9
900 psi 51 80.4
Part B: The construct used was PHP18644. Bombarded small embryos
were cultured in NLN-13S for 2-3 weeks. The embryos or excised hypocotyls from
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the embryos were cultured in bud regeneration medium MMW + IAA +TDZ +
kanamycin (50 mg/I) (embryos) or MMW + BAP + Kanamycin (50 mg/I) (for
hypocotyls).
Table 2 shows the results of resistant shoot formation using different rupture
disk strengths. In part B of the experiment, no significant difference was
found
using the 650 psi, 900 psi or 1100 psi rupture disks. The 650 and 900 psi
rupture
disks were easiest to use because less time was required to achieve the
pressure
to rupture the disks. Accordingly, the 650 psi and 900 psi rupture disks were
used
in later experiments. Table 2 also shows that excising hypocotyl segments from
the bombarded embryos results in resistant shoot formation.
Table 2. Effect of rupture disk on resistant bud production from bombarded
microspore-derived embryos
Rep # Rupture # hypocotyls or Resistant buds (%)
disks (psi) # Embryos
Rep 1 650 300 (E) 0(0.0)
900 350 (E) 0 (0.0)
1100 450(E) 1(0.2)
Rep 2 650 150 (H) 2(1.3)
1100 150(H) 0(0.0)
Rep 3 650 400 (E) 0(0.0)
900 400 (E) 1 (0.3)
1100 400(E) 0(0.0)
Rep 4 650 850 (H) 5(0.6)
900 525 (H) 3 (0.6)
1100 775 (H) 3(0.4)
Experiment 2. Resistant shoot production from bombarded microspore-
derived embryos in solid kanamycin selection medium and confirmation of
the kanamycin resistant plants by polymerase chain reaction (PCR) analysis
The objective was to demonstrate that kanamycin resistant plants were
produced from bombarded microspore-derived embryos. The construct used was
Z0 Pi~P~i654~ and the cultivar was 46A65. Kanamycin resistant shoots were
produced by bombardment of microspore-derived embryos. The shoots were
regenerated into kanamycin resistant plants. The kanamycin resistant plants
were
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analyzed using the REDExtract-N-AMPT"' plant PCR kit from Sigma as is known to
those skilled in the art. DNA was extracted from 14 kanamycin resistant plants
and
analyzed by PCR for the nptll gene. Table 3 indicates that the nptll gene was
found in 12 of the 14 plants. Accordingly, stable transgenic plants were
obtained
by bombarding microspore-derived embryos.
Table 3. Detection of nptll gene in kanamycin resistant plants regenerated
from
bombarded microspore-derived embryos
Rep # Events PCR-nptll positive
Rep 1 9 7
Rep 2 5 5
Total 14 12
Microspore-derived hypocotyls:
Experiments 3 to 7 describe work done using microspore-derived
hypocotyls. Microspores were isolated as is known to those skilled in the art
and
cultured in NLN medium for 21 to 28 days (Swanson et al., 1987). Hypocotyls
were
excised from the embryos produced from the microspores when the embryos were
approximately 3-5 mm in size.
Microspores of cultivar 46A65 were cultured in NLN medium for
approximately 21 days. On the 21st day, the NLN medium was changed and
diluted (1:20) with fresh NLN medium and the embryos were cultured for 5 more
days. The hypocotyls were excised from the embryos and preconditioned
overnight on MMW + BAP (4 mg/I) + NAA (0.25 mg/I). The hypocotyls were
transferred to osmotic medium (for example, 17% sucrose + 1 lo agar + 1 g/l
MES,
pH 6.0) for 4 hours and then bombarded. The DNA construct used in
bombardments was PHP18644. After bombardment, the hypocotyls were cultured
on the osmotic medium for approximately between 4 and 20 hours (for example,
o ~er~~igi~t), o he uorrii~ardea hypocotyis were then cultured on MMW + BAP (4
mg/I)
+ kanamycin (25-50 mg/I) for bud induction. Regenerated buds were cultured on
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MMW + kanamycin (50-100 mg/I) or MMW + BAP (0.2 mg/I) + kanamycin (50-100
mg/I) for plant regeneration.
The following experiments were done to test the effect of sucrose
concentration, amount of gold particle used and the effect of rupture disk on
transformation frequency.
Experiment 3: Effect of sucrose concentration in osmotic medium on
transient GUS expression on microspore-derived hypocotyls
The objective was to find the appropriate concentration of sucrose in the
osmotic medium (sucrose + 1% agar + 1 g/I MES, pH 6.0) in order to produce the
highest transformation efficiency.
Sucrose concentrations of 15, 17, 19 and 21% were tested as shown in
Table 4. The results indicated that using 19% sucrose produced the greatest
number of transiently transformed cells.
Table 4. Effect of sucrose concentration in osmotic medium on transient GUS
expression in microspore-derived hypocotyls
Sucrose ( /a) # hypocotyls # with GUS # total spots # spots /hypocotyls
15 24 23 236 9.8
17 24 23 242 10.1
19 24 23 475 19.8
21 24 24 400 16.7
Experiment 4. Determining the effect of the amount of gold used per shot on
transient GUS expression in microspore-derived hypocotyls
The objective was to determine the optimal amount of gold particles per
bombardment.
The construct used was PHP18644. Table 5 shows the results using 100,
200 and 300 iag of gold per shot. The results were not consistent in three
experiments. 100 pg gold particles per shot were used in the remainder of the
experiments.
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Table 5. Effect of the amount of gold particle per bombardment on GUS
transient
expression using microspore-derived hypocotyls
Rep # Gold amount/shot (ug) # hypocotyls # with GUS spots Spots/per hypocotyls
1 100 32 1 0.1
200 32 4 0.5
300 33 4 1.3
2 100 24 24 39.6
200 24 24 29.6
300 25 25 35.4
3 100 24 21 20.1
200 24 24 26.8
300 24 24 40.1
Experiment 5. Effect of rupture disks on GUS transient expression on
microspore-derived hypocotyls.
The objective was to determine which of four rupture disk strengths tested
produced the greatest number of cells expressing GUS.
The construct used was PHP18644. Table 6 shows the results of GUS
transient expression using a rupture disk strength of 450 psi, 650 psi, 900
psi or
1100 psi. Results indicate that using rupture disks 650 psi and 900 psi
produced
the highest number of cells transiently expressing GUS.
Table 6. Effect of rupture disk strength on GUS transient expression using
microspore-derived hypocotyls
Rep Rupture disks # hypocotyls # with GUS spots # GUS spots/hypocotyls
1 450 24 23 19.5
650 24 24 48.3
900 25 24 37.4
1100 24 22 27.5
2 650 24 24 60.7
900 25 25 60.5
1100 25 25 49.4
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Experiment 6. Second experiment to determine the effect of rupture disk on
resistant bud production using microspore-derived hypocotyls
The objective was to screen rupture disks to determine those that produced
the greatest number of resistant buds on 50 mg/i kanamycin.
The construct used was PHP18644. Table 7 shows the results using rupture
disks of 450 psi, 650 psi and 900 psi. No significant difference was found
between
rupture disks 450 psi, 650 psi and 900 psi in this experiment. \
Table 7. Effect of rupture disk strength on the production of kanamycin
resistant
shoots using microspore-derived hypocotyls as the bombardment tissue
Rep # Rupture disks # hypocotyls # with green buds
1 450 psi 375 6(1.6%)
650 psi 525 8 (1.5%)
900 psi 300 5 (1.7%)
2 450 psi 200 1(0.5 /a)
650 psi 225 1 (0.4%)
900 psi 100 1 (1.0 la)
3 450 psi 300 2(0.7 /a)
650 psi 300 1 (0.3%)
900 psi 300 2 (0.7%)
4 450 psi 300 0(0.0%)
650 psi 250 0 (0.0%)
900 psi 300 3 (1.0 /a)
5 450 psi 150 1 (0.7%)
650 psi 275 5 (1.8 l0)
900 psi 200 3 (1.5%)
6 450 psi 125 0(0.0%)
650 psi 75 0 (0.0 /a)
900 psi 25 1 (4.0%)
Experiment 7. Results of the GUS assay on the kanamycin resistant plants
The objective was to confirm the transgenic status of the kanamycin
i5 MSIJIGii_IL ~iiciiii~ uy i+S andiysis. i abie 8 shows that seven kanamycin
resistant
plants were analyzed by GUS assay. Six plants were positive. This confirms
that
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stable transgenic plants were produced by bombarding microspore-derived
hypocotyls.
Table 8. Confirmation of transgenic status of kanamycin resistant plants from
microspore-derived hypocotyls by GUS analysis
Rep # Resistant Plants GUS Positive Plants
Rep 1 2 2
Rep 2 3 2
Rep 3 1 1
Rep 4 1 1
Pre-incubated microspores:
Experiments 8 to 17 describe the work done using pre-incubated
microspores. The microspores were cultured in NLN-17S/10S for 2-10 days.
Optimally, the microspores are cultured for 5-7 days. Embryos could not be
detected with the naked eye. The embryogenic microspores were collected so
that
they remain viable and embryogenic with a NitexTM sieve (15-36 um in pore
size) or
Percoll (35-45%) gradient centrifugation.
Pre-incubated microspores were used as bombarded materials. Any
Brassica line that is capable of regenerating by microspore culture can be
used.
The microspores were cultured for 1-3 days in NLN-17S at 31.5 C, and then in
NLN-10S for 4-5 days at 25 C. The constructs used in the bombardments were
PHP18644, PHP21965, PHP22024, PHP22021, and PHP23560 (see Table 9).
The constructs can be the full plasmid or the expression cassette only. For
example, PHP22024 can be either the expression cassette or the full plasmid.
The
pre-incubated microspores were filtrated with sieves of pore size of 151am to
36pm.
The collected microspores were used for bombardment. The microspores were
loaded on a sieve 15pm or 201am on two layers of filter-paper and dried for
less
than one minute. The microspores and sieves were transferred to osmotic medium
that contained B5 components, 1 g/l MES and 0.8-1.6% gelrite, 15-21% sucrose
' --- ~ "-_-----~.--~ ---- -- --_ -z_-, w.....
f~!J
=-~--- on '= -- -~IIIUI-=-
n~=
-'~. TL,~ - :~~-- ~_~'~- '_- - _-_- _ ~ ~ .
-- --
\i -. . .. " vl ' e se~ t~~t'viv3 c~ vv~,ic ucc.~~cu ivi ~ii occaal VIIC IIVuI
Vtl Ule VII
medium before bombardment. The pre-incubated microspores were bombarded
with 12.5 ng to 5 pg DNA per preparation, 15-100 pg Au particles per shot, 2.5
M
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CaCI2, and 650-900 psi rupture disk. During bombardment a surface of the pre-
incubated microspores was exposed to the path of the bombarded particles
coated
with DNA (i.e. the surface was not embedded in the medium). The bombarded
microspores were cultured at least four hours in the osmotic medium after
bombardment. One to two sieves holding the microspores were cultured in 5 ml
of
NLN-1 3S with or without glyphosate per plate for approximately 7 days in the
dark.
After the 7 days, the spent medium was replaced with 10 ml of NLN-6.5S in each
plate or the spent medium was diluted to obtain a sucrose concentration 6.5%
in
each plate. If the selectable marker gene was the NPTII gene, NAA, BAP and
G418 were added to the medium. If the selectable marker gene was GAT,
glyphosate was added in the medium. In this medium containing 6.5% sucrose,
the
embryos were cultured under dim light of approximately 240 foot-candles or
2,583
lux. The final concentration of G418 was 10 mg/I. and the concentration of
glyphosate was 0.1 mM or 0.2 mM. A doubling agent was optionally added to the
medium. Resistant embryos were recorded after two to three weeks of culture.
PHP18644 and GAT (glyphosate acetyitra n sfe rase) constructs (see Table 9)
were used in transformation experiments using pre-incubated microspores. The
GAT gene was isolated from a bacterium as described by Castle et al. (2004)
Science 304: 1151-1154. The gene was shuffled 11 rounds for increasing
expression level of glyphosate acetyltransferase. There was one to several
variants in each shuffiing round. PHP18644 also contains the GUS marker gene.
The constructs are described in Table 9.
Table 9. Constructs
Constructs Promoter Selectable Marker Variants and rounds
PHP18644 CaMV NPT11 NA
PHP22024 H2b GAT GAT4604, R 8
PHP21965 SCP1 GAT GAT4604, R 8
PHP22021 H2b GAT GAT4618, R 11
PHP23560 SCP1 GAT GAT4621, R 11
For a detailed description of the protocol, see section labeled "Protocol"
following the Examples.
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WO 2007/055687 PCT/US2005/040646
Experiment 8: Development of a selection kill curve using Geneticin (G418)
(Sigma G8168)
The objective was to determine the concentration of G418 that kills non-
transgenic microspores.
Prior to this experiment, selection was done on solid medium, not in liquid
medium. Using liquid selection for Brassica transgenic cells is novel.
Selection in
liquid medium is advantageous for at least the following reasons: (a) it
allows
selection at an early stage, thereby eliminating the need for subsequent
transfers of
explants, (b) it allows for a cleaner selection because the explants are
generally
smaller when they are in liquid medium and the liquid allows all the surfaces
of the
explant to be exposed to the selection agent, (c) it may reduce the frequency
of
chimeras, and (d) a lower amount of the selection agent is needed therefore
reducing the toxicity to the researcher and the environment. The effect of
various
concentrations of kanamycin on embryo growth in liquid medium was tested. The
result showed that 10 mg/I kanamycin was sufficient to bleach non-bombarded
embryos and inhibit non-bombarded embryo growth. However, kanamycin at 10
and 20 mg/I faiied to inhibit the growth of the bombarded embryos, although
the
embryos were pale green and purple in color. Accordingly, it was difficult to
discriminate transgenic tissue or embryos from non-transgenic. Replacing
kanamycin with 5-10 mg/I G418 in NLN with 6.5% sucrose medium, resulted in a
cleaner selection. Table 10 shows that 5 to 10 mg/I G418 is sufficient to
differentiate transformed cells from untransformed cells.
38
CA 02629284 2008-05-09
WO 2007/055687 PCT/US2005/040646
Table 10. Effect of G418 on embryo growth and survival
Conc. Of G418 # embryos # Green embryos or with green island
(mg/I)
0 30 30
30 30
1.25 30 30 (paler)
28 28 (paler)
2.5 25 14 (light green, smaller size)
25 11 (light green, smaller size)
25 0
25 0
30 0
30 0
Experiment 9: Production of G418 and glyphosate resistant tissue and buds
after bombardment of pre-incubated microspores followed by selection in
5 liquid medium containing G418 or glyphosate.
The objective was to obtain transgenic plants by bombarding pre-incubated
microspores that were cultured for up to 11 days and selecting resistant
tissue and
buds on liquid medium supplemented with 10 mg/I G418 or glyphosate at 0.1mM
and 0.2mM.
10 In a first set of experiments, seven, eight, nine and eleven day old pre-
incubated microspores were bombarded with PHP18644. NAA (0.5 mg/I) and BAP
(0.05 mg/I), both growth regulators, were added to the selection medium
comprising 10 mg/I G418 for enhanced cell growth conditions. Resistant
microspores were transferred and cultured in the selection medium
MMW+IAA+TDZ+AgNO35+C+K100 to induce resistant buds and confirm
resistance. Accordingly, selection was initiated in the liquid culture medium
with
G418 and completed in the solid culture medium using kanamycin.
In a second set of experiments, 3, 4 and 5 day old pre-incubated
microspores were bombarded with PHP23560. Glyphosate was added to the liquid
and solid medium for selection.
Table 11 shows the results of the experiments. The data indicate that
bombardment of pre-incubated microspores followed by selection in liquid
medium
using G418 or glyphosate produces resistant buds and PCR positive shoots.
39
CA 02629284 2008-05-09
WO 2007/055687 PCT/US2005/040646
Table 11. Effect of the pre-incubation period on resistant bud production
Expt # # days of Embryos Resistant buds PCR confirmed
culture (%) shoots
1 9 2399 0(0.0) NA
2 11 5629 0(0.0) NA
3 8 1297 4(0.3) NA
4 7 1998 4(0.2) NA
5 NA NA 6
6 4 NA NA 3
7 3 NA NA 3
Experiment 10. Determining the effect of osmotic culture medium after
5 bombardment on resistant embryo production
The objective was to determine whether culturing bombarded tissue on
osmotic medium after bombardment would increase transformation efficiency.
Table 12 indicates that bombarded pre-incubated microspores cultured on
osmotic medium for 4 hours after bombardment produced a greater number of
transgenic sectors than bombarded pre-incubated microspores that were not
cultured on osmotic medium soon after bombardment. After the osmotic
treatment,
the bombarded pre-incubated microspores were cultured on liquid NLN medium.
Table 12. Effect of osmotic culture after bombardment on resistant embryo
production
Treatments # of embryos # with green islands
Culture 0 hour 2329 0(0.0 l0)
after bombard
Culture 4 hour 5057 80 (1.6%)
after bombard
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WO 2007/055687 PCT/US2005/040646
... ... .....
Experiment 11. Determining the efficiency of resistant bud production from
bombarded pre-incubated microspores selected on liquid medium
supplemented with G418 at 10mg/I and confirmation of resistant plants by
PCR analysis.
The objective was to confirm that selection in liquid medium produces
resistant buds.
The construct used was PHP18644. Embryos that were resistant in the
liquid selection medium were cultured in MMW + IAA (0.25-4 mg/I) + TDZ (1
mg/I) +
AgNO3 (5 mg/I). Resistant buds were isolated and cultured in B5 + GA +
kanamycin (100 mg/I) or MMW + kanamycin (100 mg/I) to produce shoots. Table
13 shows that resistant buds were regenerated from the embryos with sectors of
resistance. Accordingly, selection in G418 liquid medium is efficient.
Table 13. G418 liquid medium selection efficiency in terms of resistant bud
production.
Rep # # green embryos or islands # embryos with resistant buds
Rep 1 51 40
Rep 2 25 17
Rep 3 1 1
Rep 4 26 11
The nptll gene was confirmed in resistant plants using PCR analysis. Eight
plants were selected at random and analyzed, six were found to have the nptll
gene (Table 14).
Table 14. PCR analysis of resistant plants
Reps Resistant Plants PCR-nptll positive
Rep 1 1 1
Rep 2 1 1
Rep 3 2 1
Rep 4 4 3
Tntal R ~
1 ' I f - f
41
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WO 2007/055687 PCT/US2005/040646
Experiment 12. Glyphosate resistant embryo selection after bombarding pre-
incubated microspores with PHP22021 and PHP22024 and followed by
selection in liquid medium NLN-6.5S containing 0.1 mM and 0.2 mM
glyphosate
The objective was to compare the effect of glyphosate concentration on
selection efficiency. Table 15 shows that using either 0.1 mM or 0.2 mM
glyphosate allowed identification of resistant embryos. Both normal and
abnormal
embryos were produced. Although transgenics were identified using both 0.1 mM
and 0.2mM glyphosate, selection at 0.2mM produced fewer false positive
results. It
was more efficient and eliminated additional transfers.
42
CA 02629284 2008-05-09
WO 2007/055687 PCT/US2005/040646
ia
~
0
c
O r O O O O O
v M O 00 N U') M
N
0
O~ E + '+
d ~
U)
co
0
~
E
a) a
C ~
cc (0 r 00 O) r- 'Cr U')
~ E M O O O ~ O (V O C7
(n a) 0
C
C y
O p
ui E
O ~
>+
~ M
4- ~f
O
C
O (0 (0 rn rn 00 00 ti (0 oo 00
+N
O
U
N
o 2 2 2 2 2 ~
E E E E E E E E E E
U !1f r r N r= N r' N r'= N r N
0 O O O O o O O O O O
W =
Q
Z+
LO
N M tf) CO
~ r s- r Q' cj' ~1'
N N N N N N
O O O O O O
N a a a ~
z z z z z z
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L (a
a
CA 02629284 2008-05-09
WO 2007/055687 PCT/US2005/040646
Experiment 13. Determining the effect of sucrose concentration in the
osmotic medium on resistant embryo production
The purpose of this experiment was to find the optimal concentration of
sucrose in the osmotic medium.
Microspores were cultured for 4-7 days using 17S/10S protocol. The
construct used was PHP23560 and the concentration of DNA was 28
ng/preparation. The rupture disk was 900 psi. The bombarded microspores were
cultured in the dark for 7-10 days in NLN-13S containing a doubling agent and
0.2
mM Glyphosate. The culture was diluted with NLN-OS without glyphosate to NLN-
6.5S containing 0.1 mM glyphosate. The diluted cultures were incubated in the
light for 2-3 weeks. There was no significant difference in the number of
green
embryos (normal and abnormal) produced using 15%, 17% and 19% sucrose.
The experiment was repeated as shown in Table 16. The results of this
experiment showed that a sucrose concentration of 17% produced similar result
as
a sucrose concentration of 19% in four experiments.
Table 16. Effect of sucrose concentration in the osmotic medium on resistant
embryo production
Rep # Sucrose concentration # of plates Resistant
embryos/plate
1 17% 8 2.3
19% 8 1.8
2 17% 1 0
19% 6 1
3 17% 6 2.7
19% 6 1.8
4 17% 7 5.1
19% 8 3.9
Total 17% 2.5
19% 2.1
44
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WO 2007/055687 PCT/US2005/040646
Experiment 14. Determining the effect of the duration of the osmotic
treatment on resistant embryo production
The objective was to determine whether a 4 hour or 20 hour culture in
osmotic medium results in a greater number of transgenic events.
The construct used was PHP21965. Microspores were pre-incubated for 6
to 7 days, and then collected with 25 uM NitexTM sieve. The collected
microspores
were subsequently loaded onto NitexTM sieves with pore size of 20 uM. The
bombarded microspores and NitexTM sieves were cultured for 4 hours or 20 hours
on osmotic medium B5 + I g/I MES + 190 g/l sucrose + 12 g/l geirite (pH 5.8-
6.0)
after bombardments. Table 17 shows that treating microspores for 4 hours
produced a comparable number of resistant embryos to treating microspores for
20
hours.
Table 17. Effect of the duration of the osmotic treatment on resistant embryo
production
Rep # Osmotic duration # bombardments # resistant embyros
1 4h 8 7
44 h 4 1
2 4h 8 8
h 8 11
3 4 h 8 20
20h 8 4
4 4h 8 7
20 h 8 10
5 4 h 8 27
20 h 8 40
Summary 4h 41 69 (1.7/bombardment)
66 (1.8/bombardment)
20 h (including 44 h) 37
Experiment 15. Determining the effect of the rupture disk strength on
resistant embryo production
~
T~ ; Nu; pose ;>> ie IR-3 c;+ij.:ie~ io iei it vvcts ~u ii3i iipdre ei ieGt o~
lnbl1 psi and -''~'Uu psi
20 rupture disk strength on resistant embryo production. Construct PHP23560
was
used for bombardments. A total of nine experiments were conducted to compare
CA 02629284 2008-05-09
WO 2007/055687 PCT/US2005/040646
rupture disks of 650 psi and 900 psi strength. No significant difference was
found
between rupture disks of 650 psi and 900 psi strength (Tabie 18). The rupture
disk
650 psi was easier to use than the 900 psi rupture disk because less time was
needed to build up helium pressure.
Table 18. Effect of rupture disk strength on resistant embryo production
Rep # Rupture disks # bombardments # resistant embryos
1 650 8 21
900 8 17
2 650 9 1
900 4 5
3 650 8 10
900 7 20
4 650 8 16
900 8 13
5 650 8 8
900 8 5
6 650 8 6
900 8 3
7 650 8 0
900 8 1
8 650 8 1
900 8 3
9 650 7 4
900 6 2
Summary 650 72 67 (0.93/bombardment)
69 (1.06/bombardment)
900 65
Experiment 16. PCR analysis to confirm transgenic plant production via
bombarding pre-incubated microspores
One hundred twenty-five resistant plants were confirmed positive using PCR
analysis in 33 transformation experiments. Each experiment, on average,
produced 3.8 transaenic: r~lants (Tab(P 1c~)_ The data for pNp'?n?! ~~-d
~o??n?~
includes data produced using both the DNA expression cassette and the entire
plasmid. For example, of the 13 experiments listed under PHP22024, 9 were done
46
CA 02629284 2008-05-09
WO 2007/055687 PCT/US2005/040646
using the plasmid and 4 were done using the expression cassette. The number of
GAT positive plants was 55 and 34 respectively.
Table 19. Results of the PCR analysis of resistant plants.
Plants # GAT
Constructs # Experiments analyzed positive
PHP22024 13 89 89
PHP21965 3 8 7
PHP22021 17 31 29
Total 33 128 125
Experiment 17. Transgene copy number analysis in GAT transgenic plants.
Transgenic (T1) plants identified by glyphosate resistance were analyzed
using Southern blot hybridization analysis. Plant genomic DNA was extracted
using cetyltrimethylammonium bromide (CTAB) buffer (Rogers et al., (1994)
Plant
Molecular Biology Manual, 2"d Ed. 1:1-8). The DNA samples were digested with
Bam HI or Pst I. The hybridization probe was the GAT gene. The hybridization
was made following Rajasekaran et al. (2000) Plant Cell Rep. 19: 539-545. The
GAT gene copy number was determined by the highest number of bands from the
hybridization blots for each digest. For example, if only one band was
produced
from both Bam HI and Pst I blots for an event, it indicated that the event had
one
copy of the GAT gene. Results from 42 events showed 17 events or 40% had only
one copy of the GAT gene (Table 20).
47
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Table 20: Transgenic copy number analysis in GAT transgenic plants
Total Copy number of the GAT gene
number determined by Southern blot
Constructs of events hybridization analysis
1 2 3 >3
PHP22021 11 8 1 0 2
PHP22024 29 8 11 7 3
PHP21965 2 1 1 0 0
Total 42 17 13 7(17%) 5
(40%) (31%) (12%)
All publications and patent applications mentioned in the specification are
indicative of the level of those skilled in the art to which this invention
pertains and
are incorporated by reference.
Although the foregoing invention has been described in some detail by way
of illustration and example for purposes of clarity of understanding, it will
be
obvious that certain changes and modifications may be practiced within the
scope
of the appended claims.
Protocols
Production of pre-incubated microspores
Microspores are cultured as is known to those skilled in the art, for example
see Fukuoka et al. (1996) Plant Physiol. 111:39-47; Keller et al. (1987) Proc.
7t" Int.
Rapeseed Congr. (Plant Breeding and Acclimatization Institute, Poznan, Poland)
pp. 152-157, Swanson et al. (1987) Plant Cell Reports 6: 94-97 and Baillie et
al.
(1992) Plant Cell Reports 11: 234-237. A detailed procedure is provided as
follows:
Collect about 400 buds at uni-nucleus microspore stage from a Brassica
variety that is responsive to microspore culture and regeneration, for example
46A65, Westar or Topas. Sterilize the buds in 5% sodium hypochlorite solution
(100% commercial bleach solution) and let sit for 15-20 minutes. Place the
buds in
sterile water for 5 minutes to rinse off bleach. Repeat this step two more
times.
EmlJtv buds into blender c~ ~nG ?nd blend f~!r 8 cA~'s?'"~ds '~t 4o~-"--' ~?''-
-~~-'~ in 'M - '--'q -I
- -. _ - - _ -~., ~ . ., ~v ,,.,
B5-W. Filter contents through two nested 44 pm Nitex filters into 50m1
centrifuge
tubes. Wash filters with 20 - 25 ml B5-W, cap tubes and centrifuge at about
1,000
48
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revolutions per minute (rpm) for 6 minutes. Decant B5-W, add 45 ml B5-W,
centrifuge, decant, add 45 ml B5-W, centrifuge and repeat for a total of 4
washes.
Before plating microspores, adjust microspore density to 100,000 microspores
per
ml with NLN-17S using a heamocytometer. Plate microspore suspension in 9-cm
plates at 6 mI/plate. Culture the plates (around 40) for 2-3 days at 31.5 C,
followed
by NLN-10S. Culture the plates at 25 C for an additional 3-4 days.
Particle bombardment
Methods of particle bombardment are known to those skilled in the art.
Detailed directions and procedures are provided upon purchasing a particle
gun.
For example, see Manual of Biolistic PDS-1000/He Particle Delivery System. For
additional references, see Sanford et al., U.S. Patent No. 4,945,050; Tomes et
al.,
U.S. Patent No. 5,879,918; Tomes et al., U.S. Patent No. 5,886,244; Bidney et
al.,
U.S. Patent No. 5,932,782; Tomes et al. (1995) "Direct DNA Transfer into
Intact
Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ
Culture: Fundamental. Methods, ed. Gamborg and Phillips (Springer-Veriag,
Berlin); and McCabe et al. (1988) Biotechnology 6:923-926. A detailed
procedure
is provided below:
Make gold particle aliquots
Use 250 mg gold particles with diameter of 0.6 micron. Add 1000 ial EtOH
and sonicate 5-8 seconds. Divide gold suspension to two tubes (500 pl each)
(Fisher 05-541-27). Centrifuge for one minute, 13,000 rpm and pipette off
EtOH.
Wash with 1 ml sterile distilled water three times. Wash gold pellets with
1000 pl
EtOH and Pipette off EtOH. Add 800 pl EtOH to each tube and suspend gold
particles. Weigh 16 tubes (Fisher 05-541-27). Aliquot 100 lal gold suspension
to
each tube. Centrifuge 30 seconds and pipette off EtOH. Dry for one hour and
weigh tube/gold to calculate gold weight in each tube. Add sterile water to
each
tube to make gold concentration to 3 mg/50 lal. Suspend gold particles with
pipette
sucking-releasing and sonication (3 seconds). Aliquot gold suspension to new
1.5
ml tubes at 50 uI/tube (3 mg/tube). Store in -20C freezer.
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Coat DNA on gold particles
Gold particle amount per shot is 100 pg/shot (3 mg/30 shots). Add 3 pg
DNA/bp/prep plasmid DNA, 50 l CaC12 (2.5 M, aliquoted into small volumes), 20
pl spermidine (0.1 M, base-free, aliquoted into small volumes) to a tube of
gold
particle aliquot. Pipette 30-50 times after each addition. Shake for 3 minutes
on
vortex shaker. Centrifuge for 10 seconds at 10,000 rpm and discard
supernatant.
Gently add 200 lal 100% EtOH and set in ice for 10 minutes and discard
supernatant. Wash gently with 200 pI 100% EtOH twice and discard washes. Add
150 pl 100% EtOH. Use 10 pl pipette tip to suspend the gold pellet. Fully
suspend
gold particles. Sonicate for 3 one-second dips to break small pellets.
Sterilize
macro-carrier discs in 70% EtOH for minimum 10 minutes. Transfer to 100% EtOH
then dry in laminar flow cabinet. Place 5 pl DNA-coated gold particle
suspension
on the centre of each sterilized macro-carrier disc and dry for at least half
an hour.
Biolistic gun operation
The microcarrier launch assembly parts, the macrocarrier holders, the
rupture retaining cap, macroccarrier, petri dish holder and the stopping
screens can
be sterilized either by soaking (or spraying) in 70% EtOH for 15 minutes and
drying
in the laminar flow cabinet, or by autoclaving. The rupture disks should be
sterilized in 50% iso-propanol for 10-30 seconds. Sterilize the chamber by
spraying
70% EtOH. Build up helium pressure higher than rupture disk. Load the rupture
disk retaining cap and microcarrier launch assembly and assemble in chamber.
Place the microcarrier launch assembly in the first slot from the top.
Position the
sample on the petri dish holder at the third slot. Close the chamber. Set the
vacuum switch on the gun to VAC position. Once the vacuum level is reached to
or
beyond 27 inch Hg, place the vacuum switch in the HOLD position. Press and
hold
FIRE switch until burst. Release the fire switch immediately and switch the
VACUUM to the VENT position. After the vacuum is released, take out sample and
microcarrier launch assembly. Repeat until all samples are bombarded.
Bombarcirr-ent and seiection of pre-incubated microspores
Culture microspores for 6-8 days using NLN-17S/10S protocol. Collect
microspores with Nitex nylon sieve with 25 pm in pore size. Transfer
microspores
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to a piece of Nitex nylon sieve and blot extra medium with filter papers.
Transfer
microspores and the Nitex to osmotic medium (B5 + 17% sucrose + I gll MES +
0.8% gelrite, pH: 6.0) and treat at least one hour. Bombard samples with
rupture
disk 650 psi or 900 psi. Leave samples in the osmotic medium for at least 4
hours.
Transfer microspores and Nitex to NLN-13S. A chromosome doubling agent can
be added. Medium contains no selection agent (nptll selection) or 0.1-0.2 mM
glyphosate (GAT selection). Each plate (9 cm) contains 5 ml medium and one
piece of microspores/Nitex. Culture the bombarded microspores for 7 days at
25C
in dark. Replace medium with 10 ml of NLN-6.5S with final concentration of
NAA0.5BAP0.05 + G418 (10 mg/I) if using the nptll gene as selectable marker or
add 5 ml of NLN-OS with Glyphosate 0.1-0.2 mM if using the gat gene as
selectable
marker. Culture under light for 2 weeks.
Plant regeneration
Culture green embryos or tissue in MMW+IAA2+TDZO.5+STS6 with 25 mg/I
kanamycin or 0.1 mM glyphosate for 4 weeks (STS6 is silver thiosulfate at
concentration of 6 pM). Isolate regenerated buds and culture in MMW+BAPO.2 or
B5+GA with 50 mg/I kanamycin or 0.1 mM glyphosate. Excise shoots and transfer
to rooting medium 1/2MMW+1%sucrose+IBA2 with 25 mg/I kanamycin or B5+GA
with 0.1 mM glyphosate.
Transfer resistant plants to soil and PCR assay
Transfer resistant plants to soil in 36-cell flats after root is well
developed.
To maintain humidity, place lid on top for one week or until shoots have
established
themselves in soil. Assay for transgenics by PCR analysis using a PCR kit from
Sigma (REDExtract-N-Amp Plant PCR kit). Transgenic plants must be labeled as
"transgenic" or "GMO".
GUS assay
GUS analysis is known to those skilled in the art. The protocol can be found
in
numerous references, for example Wu H, McCormac AC, Elliott MC, Chen DF
(1998) Agrobacterium-mediated stable transformation of cell suspension
cultures of
barley (Hordeum vulgare). Plant Cell Tissue and Organ Culture 54:161-171.
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PCR analysis of the GAT and NPTII genes
PCR analysis is known to those skilled in the art. The protocol can be found
in
numerous references. For example, PCR analysis of the nptll gene was done
according to Broothaerts W, Wiersma PA, Lane WD (2001) Multiplex PCR
combining transgene and S-allele control primers to simultaneously confirm
cultivar
identity and transformation in apple. Plant Cell Rep 20:349-353.
Plant DNA was extracted following SigmaTM Technical Bulletin Code MB-850
and using REDExtract-N-AmpTM Plant PCR kit. The temperature cycle was 95 C, 2
min; (94 C, 30 s; 64 C, 30 s; 72 C, 30 s) for 35 cycles; 75 C, 5' for the GAT
gene
amplification, or 95 C, 2 min; (95 C, 15 s; 60 C, 30 s; 72 C, 30 s) for 35
cycles;
75 C, 5' for the nptll gene.
Product size
GAT4604 and 4618: 317 bp; GAT4621: 255 bp; NPTII: 700 bp.
PCR reaction
Reagents Volume
Water 5.2 pl
REDExtract-N-Amp PCR reaction mix 10 pl
Primer-F (10 pM/pl) 0.4 pl
Primer-R (10 pM/pI) 0.4 pl
Leaf disk extract 4 pl
Total volume 20 l
Southern blot hybridization analysis
Southern blot hybridization analysis is known to those skilled in the art. See
for
example, (see Rajasekarran et al. (2002) Plant Cell Rep. 19:539-545.
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Media recipes
MMW
MS salts and organics (Murashige and Skoog 1962 Physiol. Plant. 15:473-479)
Sucrose (3%)
MES (2 g/1)
Sigma agar #1296 (0.6%) pH 5.8
MMW + IAA + TDZ + AgNO3 + Gly
MMW
IAA (2 mg/I)
TDZ (0.5 mg/I)
silver nitrate (5 mg/I)
glyphosate (0.1 mM)
MMW + BAP + Kan 25-50
MMW
BAP (4 mg/I)
Kanamycin (25-50 mg/I)
MMW + kan 50-100
MMW
Kanamycin (50-100 mg/1)
B5
B5 vitamins and minerals (Gamborg et al. (1968) Exp. Cell Res.50:151-158)
Sucrose (2%)
Sigma agar (0.6%)
pH 5.8
B5+GA+GIy
B5
GA3 (0.1 mg/I)
Glyphosate (0.1 mM)
J..%
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B5-W
B5 (no agar)
Sucrose (130 g/1)
NLN
Components are as Lichter (1982) Z Pflanzenphysiol 105:427-434 without potato
broth and plant growth regulators. Medium pH is 6Ø
NLN-17S, NLN-IOS, NLN-6.5S
NLN contains 17%, 10% or 6.5% sucrose
Osmotic medium for pre-incubated microspores
B5
Sucrose 170g/I
MES (1g/1)
Phytagel (8-16 g/I)
pH: 6.0
Rooting medium (1/2MMW +1% sucrose+2 IBA)
Half strength MMW
Sucrose (10 g/1)
IBA (2 mg/I)
*Trade-mark
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Baim et al. (1991) Proc Natl. Acad. Sci. USA 88:5072-5076
Ballas et al., (1989) Nucleic Acids Res. 17: 7891-7903
Barkley et al. (1980) in The Operon, pp. 177-220
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