Note: Descriptions are shown in the official language in which they were submitted.
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TRANSFORMATION OF DICOTYLEDONOUS PLANTS BY AN ISOLATED SHOOT APEX MERISTEM
METHOD
TECHNICAL FIELD
The present invention relates to a method for transforming plants. In
particular,
the present invention relates to a method of transforming dicotyledons via
isolated shoot
apices.
BACKGROUND TO THE INVENTION
Transgenesis, or genetic transformation, allows for the generation of plants
with
improved traits significantly faster than conventional breeding practices.
This technology
is based on the delivery of genes of interest, from a broad range of sources,
into the plant
genome. Transformation of dicots is primarily performed utilizing
Agrobacterium-
mediated DNA delivery which relies on the ability of Agrobacterium tumefaciens
to
transfer a portion of its DNA, called T-DNA, into plant cells.
For various reasons, Agrobacterium-mediated transformations generally have
relatively low success rates. For example, there is a high potential for
somaclonal
variation in the resultant transforms. Also, certain commercially important
cultivars are
recalcitrant to transformation by Agrobacterium.
It has been suggested that a way of overcoming some of the issues with
Agrobacterium-mediated transformation would be to excise shoot apex tissue
from the
target plant and transform this tissue with an Agrobacterium vector. Shoot
apex tissue
permits rapid propagation of transformed plants as most dicots can be
regenerated into
intact plants from the shoot apex explants. See, for example, US Patent
Numbers
5,164,310; 7,122,722; 6,858,777; and US Published Patent Application Number
2003/0208795.
The shoot apex comprises meristematic tissue which contains undifferentiated
cells (meristematic cells) and is found in zones of the plant where growth
occurs. Apical
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meristems (or growing tips) are found in the buds and growing tips of roots
and shoots in
plants. An apical meristem is usually a dome-shaped structure that comprises
several
layers. The number of layers varies according to plant type. In general the
outermost
layer is called the tunica and is comprised of epidermal (L 1) and sub-
epidermal (L2)
layers. The innermost layers (L3) are known as the corpus. The corpus and
tunica play a
critical part in plant physical appearances as all plant cells are formed from
the
meristems. Cells in the L1 and L2 layers divide in a sideways fashion which
keeps these
layers distinct, while the L3 layer divides in a more random fashion.
Brassica species include a large group of agriculturally important crops
including
canola. Canada and the United States produce between seven and ten million
tonnes of
canola seed per year. Annual Canadian exports alone total three to four
million tonnes of
the seed, seven hundred thousand tonnes of canola oil and one million tonnes
of canola
meal. Canola oil has been claimed to promote good health due to its very low
saturated
fat and high monounsaturated fat content, and beneficial omega-3 fatty acid
profile.
Canola was developed through conventional plant breeding but the cultivars
grown today
are mostly varieties that have been genetically engineered. For example,
approximately
80% of canola planted in Canada has been modified to be herbicide-tolerant.
However,
some of the most commercially important brassica cultivars are recalcitrant to
Agrobacterium transformation.
SUMMARY OF THE INVENTION
The present invention relates to a method of transforming dicotyledonous
plants
with a vector comprising the genetic material of interest. The present method
comprises:
(a) isolating the shoot apices from seedlings and optionally, culturing the
isolated apices on an appropriate medium;
(b) dividing a selected shoot apex into at least two, preferably asymmetric,
pieces so that a portion of the apical meristem is exposed;
(c) making an incision into the meristematic region of the apical meristem;
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(d) exposing the incised meristematic region to a transformation vector; and
(e) optionally, culturing the incised shoot apex.
The present method provides effective transformation of dicotyledonous plants
with a high frequency of success and reduced incidence of somaclonal
variation. The
present method permits regeneration of transformants in a reasonable time
period. The
present method can be performed without using selection media which can
negatively
impact the regeneration efficiency in tissue culture.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be described in conjunction with reference to the
following drawings, in which:
Fig. 1 shows a schematic representation of a typical apical meristem;
Fig. 2 is a micrograph of a Brassica sp. shoot tip; and
Fig.3 shows a schematic representation of a shoot tip.
DETAILED DESCRIPTION OF THE INVENTION
The present method comprises isolating shoot apices from seedlings by, for
example, excision. Preferably the seedlings are at least three days old, more
preferably at
least five days old, even more preferably at least seven days old. Preferably,
the seedlings
are fifteen days old or less, more preferably ten days old or less.
The isolated apicies are preferably incubated for a least one day, preferably
two
days, prior to further processing. This period of incubation may conveniently
be
performed in a culture medium such as basal Murashige and Skoog medium (MS),
Shoot
Induction Medium (SIM), Callus Induction Medium (CIM), or mixtures thereof.
Preferably, the pre-culture is done on CIM. Pre-culture of the explants seems
to enable
them to better cope with the stress of exposure to Agrobacterium.
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In the present method the apex is divided into at least two pieces. Preferably
the
pieces are asymmetrical. The division may be performed in any suitable manner
that
exposes the apical meristematic tissue. Preferably, the lateral face of the
meristem is
exposed. In one preferred embodiment, one of the cotyledonary leaves is peeled
off
lengthwise so as to expose the lateral face of meristem.
The present method comprises making at least one incision into the exposed
meristematic tissue. It is highly preferred that the incision penetrates to
and exposes the
L2 layer. Preferably, at least two incisions are made. These incisions may be
made in
any suitable portion of the exposed apical meristem but preferably they are
made on
either side of the apical dome. While not wishing to be bound by theory, it is
believed
that exposing the L2 layer improves the efficacy of the transformation method
because
the L2 layer contributes most of the mesophyll tissue of the resultant plant.
The present method comprises exposing the isolated shoot tip to a plant
transformation vector. The vector may take any form suitable for transforming
dicotyledons. It is highly preferred that the present transformation is
mediated by
Agrobacterium lumefaciens.
The shoot tip explants may be exposed to Agrobacterium harbouring the DNA of
interest by any suitable means. Preferably the explants are added to an
Agrobacterium
suspension for a suitable period of time such as 5 minutes. The suspension may
be
shaken in order to maximize the chances of a successful infection.
After exposure to the vector, the explants may then be regenerated. For
example,
the explants may be placed shoot induction medium and incubated for a suitable
period.
The explants may be transferred to selection media for selection of
transformants
therefrom. When the shoots are of an appropriate size, they may be transferred
to root
induction media for root regeneration. Rooted plants can be grown under
suitable
conditions known to those skilled in these arts.
Preferred targets for the present method include canola and other commercially
useful dicots. The present method is particularly useful for those cultivars
that are
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recalcitrant to traditional transformation methods, such as for example TOPAS;
TOPAS
4079, Zarfam, Modena, and Opera.
Referring now to the figures, Fig. 1 shows a schematic representation of an
apical
dome. An apical meristem, which includes the apical dome, is composed of the
epidermal LI layer (1), the subepidermal L2 layer (2), and the corpus L3 layer
(3).
Fig. 2 is a micrograph of a typical Brassica sp, shoot tip showing the leaf
primordia (10), the apical meristem (11), the apical dome (12), auxiliary buds
(13), and
the preferred locations for the incisions (14).
Fig. 3 shows a schematic representation of a shoot tip showing the leaf
primordial
(10), the apical meristem (11), the apical dome (12), auxiliary buds (13), and
the
preferred locations for the incisions (14).
EXAMPLE
Hypocotyl explants were obtained from 8-day-old canola seedlings (Invigor
5020)
obtained by culturing sterilized seeds in '/2 Murashige and Skoog (MS)
supplemented
with 2% sucrose, pH 5.8 (seed germination medium) with incubation under a 16/8
hour
photoperiod. The explants were then transferred aseptically on callus
induction medium
(CIM) and incubated for 2 days.
Agrobacterium suspension in MS (pH 5.6-5.8) containing 100 mM
acetosyringone was obtained by pelleting bacterial cells by centrifugation at
4000 rpm for
15 min at room temperature.
The pre-cultured shoot tip explants were dissected into two asymmetrical
pieces.
The smaller piece was discarded. An incision was made on both sides of the
meristematic
region in the larger half of the bisected explants so as to expose the germ
line cells.
Excess tissues i.e hypocotyls and cotyledonary leaves were trimmed off. Care
was taken
not to remove any shoot primordia.
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The trimmed tips were inoculated with the Agrobacterium suspension for 10 min
with constant shaking. The explants were then transferred to plates containing
CIM.
After incubation for 72 hours, the tissues were washed and transferred again
to CIM
containing 250 mg/1 carbenicillin. The explants were further incubated at 22 +
2 C under
a 16/8 h photoperiod.
After 2 weeks of incubation, the explants comprising hypocotyls were
transferred to shoot induction medium (SIM) containing 250 mg/1 carbenicillin.
Regenerated shoots were transferred to shoot elongation medium (SEM). Shoots
elongated readily in both media within 2 weeks. When the size of the shoots
reached
about 1 cm to about 1.5 cm, they were transferred to root induction medium
(RIM) for
regeneration of roots. Once rooted, the plantlets were transferred to a soil-
less growth
medium for further growth, and were checked for gene expression.
The following media were used:
Callus Induction Medium (CIM):
MS basal medium supplemented with 3% sucrose, 0.1 g/1 myo-inositol, 0.8%
agar, 1 mg/12,4-D, 5 mg/I silver nitrate, 0.5 g/l MES; pH 5.8.
Shoot induction Medium (SIM):
MS basal medium supplemented with 3% sucrose, 0.1 g/l myo-inositol, 0.8%
agar, 2 mg/1 BA, 5 mg/1 silver nitrate, 0.5 g/1 MES; pH5.8.
Shoot Elongation medium (SEM):
MS basal medium supplemented with 1% sucrose, 0.1 g/1 myo-inositol, 0.8%
agar, 0.5 g/1 MES, 0.05 mg/I BA; pH 5.8.
Root Induction Medium (RIM):
V2 MS basal medium supplemented with 1% sucrose, 0.8% agar, 0.5 g/1 MES; pH
5.8.
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