Note: Descriptions are shown in the official language in which they were submitted.
CA 02352488 2001-05-25
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PLANT TRANSFORMATION PROCESS
BACKGROUND
Genetic transformation of higher plants promises to have a major impact on
crop improvement, as well as many other areas of biotechnology. Genetic
transformation can be used to produce transgenic plants carrying new genetic
material
stably integrated into the genome and to engineer 'designer' crops with
specific traits.
Various methods of genetic transformation have been developed and applied to a
growing number of plant species. However, the ease and success rate of genetic
transformation methods varies widely among plant species [Muller, et al. 1987.
"High
meiotic stability of a foreign gene introduced into tobacco by Agrobacterium-
mediated
transformation," Mol Gen Genet 207:171-175; Gasser, C.S. and Fraley, R.T.
1989.
"Genetically engineering plants for crop improvement," Science 244:1293-1299;
Umbeck, et al. 1989. "Inheritance and expression of genes for kanamycin and
chloramphenicol resistance in transgenic cotton plants," Crop Science 29:196-
201;
Gordon-Kamm, et al. 1990. "Transformation of maize cells and regeneration of
fertile
transgenic plants," Plant Cell 2:603-618; Chabaud, et al. 1996."Transformation
of
barrel medic (Medicago truncatula Gaertn.) by Agrobacterium tumefaciens and
regeneration via somatic embryogenesis of transgenic plants with MtENODl2
nodulin
promoter fused to the gus reporter gene," Plant Cell Rep 15:305-310; Kar, et
al.
1996."Efficient transgenic plant regeneration through Agrobacterium-mediated
transformation of chickpea (Cicer arientinum L.)," Plant Cell Rep 16:32-37;
Kim, J.W.
and Minamikawa,T. 1996. "Transformation and regeneration of French bean plants
by
the particle bombardment process," Plant Sci 117:131-138; Trieu, A.T. and
Harrison,
M.J. 1996. "Rapid transformation of Medicago truncatula: regeneration via
shoot
organogenesis," Plant Cell Rep 16:6-11; Bean, et al. 1997."A simple system for
pea
transformation," Plant Cell Rep 16:513-S 19; Cheng, et al. 1997. "Genetic
transformation of wheat mediated by Agrobacterium tumefaciens," Plant Physiol
115:971-980 ; Cheng, et aI. 1997. "Expression and inheritance of foreign genes
in
transgenic peanut plants generated by Agrobacterium-mediated transformation,"
Plant
CA 02352488 2001-05-25
WO 00/37663 PCT/US99/30972
Cell Rep 16:541-544; and Tingay, et al. 1997. "Agrobacterium tumefaciens-
mediated
barley transformation," Plant) 11:1369-1376.]
The most common and widely used method of transformation of dicotyledonous
plants utilizes a bacterium, Agrobacterium tumefaciens, to effect gene
transfer.
s Agrobacterium tumefaciens is a gram-negative, soil dwelling plant pathogen
that
infects its plant host and subsequently delivers and integrates part of its
genetic material
into the plant genome. The transferred portion of DNA is termed the T-DNA
fragment,
and additional genetic material can be added to the T-DNA. The additional
genetic
material will then be integrated into the genome along with the T-DNA. In this
way,
1 o Agrobacterium can be used to facilitate the transfer of new genes into the
plant genome
(Fraley, et al. 1983. "Expression of bacterial genes in plant cells," Proc
Natl Acad Sci
USA 80:4803-4807).
While transformation with Agrobacterium has worked well for a number of
model species such as tobacco and petunia, the approach is subject to a number
of
15 limitations. Some plant species, including many monocotyledonous plant
species, are
not readily susceptible to infection by Agrobacterium (Potrykus, I. 1990.
"Gene transfer
to cereals: an assessment," BiolTechnology 8:535-542). In these cases,
alternative
approaches have been used, including particle bombardment and direct gene
transfer
into protoplasts via electroporation, microinjection, or polyethylene glycol
mediated
2o uptake [Klein, et al. 1987. "High velocity microprojectiles for delivering
nucleic acids
into living cells," Nature 327:70-73; McCabe, et al. 1988. "Stable
transformation of soy
bean (Glycine max) by particle acceleration," BiolTechnology 6:923-926;
Bornmineni,
et al. 1994. "Expression of GUS in somatic embryo cultures of black spruce
after
microprojectile bombardment," JExp Bot 45:491-495; Christou, P. 1995.
"Strategies
25 for variety-independent genetic transformation of important cereals,
legumes and
woody species utilizing particle bombardment," Eupytica 85:13-27; Kim and
Minamikawa. 1996. Plant Sci 117:131-138; Klein, et al. 1998. "Stable genetic
transformation in intact Nicotania cells by the particle bombardment process,"
Proc
Natl Acad Sci USA 85:8502-8505].
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Regardless of the method of delivery of the new genetic material, it is
necessary
to regenerate whole fertile plants from the transformed cells. The production
of stably
transformed transgenic plants involves two processes: transformation of plant
cells and
then regeneration of those transformed cells to whole plants. In most cases, a
plant
tissue explant is incubated with Agrobacterium carrying a T-DNA containing a
selectable marker gene and a 'gene of interest'. A proportion of the cells in
the explant
will become transformed, and whole plants are then regenerated from these
cells via
somatic embryogenesis or direct organogenesis. Transformants are selected by
inclusion of the appropriate selective conditions in the regeneration media.
The choice
of tissue explant depends on the plant species. Leaf, cotyledons, hypocotyls,
cotyledonary meristems, and embryos are among those that have been used
successfully.
Because neither the transformation nor the regeneration are 100% effective,
the
chance of obtaining a transformed plant depends on these two processes
occurring
t5 consecutively in the same cell. In many cases, the production of transgenic
plants is
prevented due to the inability to regenerate plants from those tissues
susceptible to
transformation. For species in which somatic embryogenesis is a viable method
of
regenerating plants, there are other limitations. Plants regenerated via
somatic
embryogenesis may show significant somatic variation, altered ploidy,
phenotypic
2o abnormalities and poor fertility (Bean, et al. 1997. Plant Cell Rep 16:513-
519). While
regeneration via direct organogenesis overcomes some of these problems, not
all plants
can be regenerated in this way. Finally, although transformation of many crop
plants is
possible, it is usually achieved in highly regenerable lines or cultivars, and
the elite
agriculturally important lines are not usually amenable to transformation.
Therefore,
25 introduction of a desired trait into the elite lines has been limited to
subsequent
traditional breeding methods following transformation of parental lines.
In order to develop a transgenic plant line expressing a new trait, it is
desirable
to produce a large number of transgenic plants from which the best expressing
line can
be selected. The requirement for a number of plant lines stems from the fact
that the
3o integration of the T-DNA fragment into the plant genome is a random event,
and
therefore, each transgenic plant will contain the new gene integrated into
different sites
CA 02352488 2001-05-25
WO 00/37663 PCT/US99l30972
of the genome. Due to this phenomenon termed 'position effect', the various
transgenic lines will vary in the levels of expression of the introduced gene
(Ulian, et
al. 1994. "Expression and inheritance pattern of two foreign genes in
petunia," Theor
Appl Genet 88:433-440). Therefore, it is desirable to produce a large number
of
transgenic lines in order to select for those expressing the introduced gene
at a high
level.
The only plant which has been successfully transformed with a high degree of
ease and efficiency is Arabidopsis thaliana, a model plant used widely for
genetic and
molecular analyses of plant developmental processes. A direct method of
1o transformation has been developed forArabidopsis thaliana (Bechtold, et al.
1993. "In
plants Agrobacterium mediated gene transfer by infiltration of adult
Arabidopsis
thaliana plants," Comptes Renus de 1'Academie des Sciences Serie 111 Sciences
de la vie
316.). In this transformation process, the plant is (1) grown to maturity, (2)
immersed
in a suspension ofAgrobacterium cells, (3) held under vacuum for a short
period of
15 time, and then (4) allowed to set seed. A proportion of the progeny is
transformed.
Recent data suggest that the gametophyte progenitor, gametophyte, or
fertilized
embryos are the targets (Bechtold, N. and Pelietier, G. 1998. "In plants
Agrobacterium-
mediated transformation of adult Arabidopsis thaliana plants by vacuum
infiltration,"
Methods Mol Biol 82:259-266). Although Bechtold's method has been tried in
other
2o species including Brassica napus and Beta vulgaris, these attempts
reportedly have
been unsuccessful (Siemens, J. Scheiler, O. 1996. "Transgenic plants: genetic
transformation-recent developments and the state of the art," Plant Tissue
Culture and
Biotechnology 2:66-75).
Leguminous crops such as peas, soybean, bean, alfalfa, peanut, chick pea,
25 pigeon pea and clover have widespread economic importance throughout the
world.
Legumes are an important source of protein as grain and forage legume crops
for
animals and as grain legumes for humans. For example, soybeans (Glycine max)
are a
major source of protein in animal and human food, and soybean oil is the most
widely
used edible oil in the world. The productivity, and therefore value, of a wide
range of
30 leguminous crops could be increased by the introduction of traits such as
disease
resistance, herbicide resistance, insect resistance, reduced levels of tannins
and lignin
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- WO 00/37663 PCT/US99/30972
(forage legumes), and improved protein and lipid quality. For example, the
soybean
cyst nematode causes losses in yield of up to one billion United States
dollars per year.
With the recent cloning of a beet cyst nematode resistance gene and a potato
cyst
nematode resistance gene (Williamson, V.M. 1999. Curr Opin Plant Biol 2:327-
31),
strategies are now being explored for genetically engineering resistance in
plants.
While there have been some attempts to introduce these traits into leguminous
crops via genetic engineering, the current transformation methods involving
tissue
culture are exceedingly labor intensive and inefficient. In particular, the
large seed
grain legumes, such as pea, bean, and soybean have proved very difficult to
transform,
to and tissues susceptible to transformation have proven difficult to
regenerate.
[Bingham. et al. 1975. "Breeding alfalfa which regenerates from callus tissue
in
culture," Crop Science 15:719-721; Hinchee, et al. 1988. "Production of
transgenic
soybean plants using Agrobacterium-mediated DNA transfer," BiolTechnology
6:915-
922; Schroeder, et al. 1993. "Transformation and regeneration of two cultivars
of pea
(Pisum sativum L.)" Plant Physiol 101:751-757; Chabaud, et al. 1996. Plant
Cell Rep
15:305-310; Kar, et al. 1996. Plant Cell Rep 16:32-37; Kim and Minamikawa.
1996.
Plant Sci 117:131-138; Trieu and Harrison. 1996. Plant Cell Rep 16:6-11; Bean,
et al.
1997. Plant Cell Rep 16:513-519; Cheng, et al. 1997. Plant Cell Rep 16:541-
544; and
Dillen, et al. 1997. "Exploiting the presence of regeneration capacity in the
Phaseolus
2o gene pool for Agrobacterium-meditated gene transfer to the common bean.
Mededelingen-Faculteit-Landbouwkundige-en-Toegepaste-Biologische-
Wetenschappen-Universiteit-Gent 62:1397-1402].
In an alternative approach, it was shown that cells within a soybean meristem
can be transformed by particle bombardment. However, this leads to chimeric
plants
with transformed sectors. Some of these sectors will eventually give rise to
seed, and
the seed will carry the transgene (McCabe, et al. 1988. BiolTechnology 6:923-
926;
Chowrira, et al. 1995. "Electroporation-mediated gene transfer into intact
nodal
meristems in planta: generating transgenic plants without in vitro tissue
culture,"
Molecular Biotechnology 3:17-23; and Chowrira, et al. 1996. "Transgenic grain
legumes obtained by in planta electroporation-mediated gene transfer,"
Molecular
Biotechnology 5:85-96). While this procedure has enabled the production of
transgenic
5
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soybean, it is very labor intensive because numerous meristems need to be
bombarded
to have a realistic chance of obtaining any transgenic seeds.
Medicago truncatula Gaertn. (barrel medic) is a diploid, autogamous, annual
medic that is grown as a pasture legume in a number of regions throughout the
world,
including Mediterranean areas, South Africa and Australia (Crawford, et al.
1989.
"Breeding annual Medicago species for semiarid conditions in Southern
Australia," Adv
Agron 42:399-437). In Australia, the annual medics are the main legume found
on over
50 million hectares of agricultural land, and a variety of species and
ecotypes have been
developed. The first commercial cultivar of M. truncatula was sown in 1938,
and this
1 o species has been favored due to its ability to tolerate both low rainfall
and high lime
soils (Crawford, et al. 1989. Adv Agron 42:399-437). Medicago truncatula also
is
emerging as a model legume for studies of the nitrogen-fixing Rhizobium/Iegume
symbiosis and the arbuscular mycorrhizal symbiosis [Cook, et al. 1995.
"Transient
induction of a peroxidase gene in Medicago truncatula precedes infection by
Rhizobium meliloti," Plant Cell 7:43-55; van Buuren, et al. 1998. "Novel genes
induced
during an arbuscular mycorrhizal (AM) symbiosis between M. truncatula and G.
versiforme," MPMI 12:171-181. The attributes that make M. truncatula a useful
model plant for molecular and genetic analyses include its small genome (4.5
times
larger than Arabidopsis), rapid life cycle, and relatively small physical size
(Barker, et
2o al. 1990. "Medicago truncatula, a model plant for studying the molecular
genetics of
the Rhizobium-legume symbiosis," Plant Mol Biol Rep 8:40-49). In addition, it
can be
transformed via Agrobacterium and regenerated via somatic embryogenesis, or
alternatively, by direct organogenesis (Thomas, et al. 1992. "Genetic
transformation of
Medicago truncatula using Agrobacterium with genetically modified Ri and
disarmed
Ti plasmids," Plant Cell Rep 11:I 13-117; Chabaud, et al. 1996. Plant Cell Rep
15:305-
310; Trieu and Harrison. 1996. Plant Cell Rep 16:6-11; Hoffmann, et al. 1997.
"A new
Medicago truncatula line with superior in vitro regeneration, transformation,
and
symbiotic properties isolated through cell culture selection," Mol Plant-
Microbe
Interact 10:307-315).
3o Although Agrobacterium-mediated transformation with regeneration via
somatic embryogenesis or direct organogenesis is a viable approach, these
methods are
6
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WO 00/37663 PCT/US99/30972
very labor intensive, not very efficient, and in some cases, very slow. While
these
approaches may be suitable for the generation of small numbers of transgenic
plants,
they cannot be used to generate the large numbers of lines required for many
genetic
approaches and high through-put systems, such as T-DNA mutagenesis or
activation
tagging.
An Agrobacterium-mediated transformation method has now been found
wherein seedlings, rather than flowering plants or tissue explants, are
utilized as the
subject biological material for exposure to Agrobacterium cells. Moreover,
following
maturation of treated plants and seed set, transgenic plants are selected
directly from a
t o population of progeny representing various insertional events. This
seedling
transformation method provides high efficiency, low labor input, and large
numbers of
transgenic plants without all the problems associated with transformation of
flowering
plants or tissue explants and regeneration via somatic embryogenesis or direct
organogenesis.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 depicts a map of the T-DNA from the binary vector pBI121-bar.
Fig. 2(a) is a Southern blot of HindIII digested DNA from transgenic plants
(progeny of infiltrated plants) hybridized with a bar probe. Samples 1.3-1.8
are from
2o Treatment 1 (I-minute vacuum infiltration); Samples 2.2-2.7 are from
Treatment 2
(1.15-minute vacuum infiltration); and Samples 3.1-3.5 are from Treatment 3
(40-
second vacuum infiltration followed by 20-second hold). The pBI121-bar plasmid
DNA is included to the left of the blot. This part of the blot was excised and
exposed
for a shorter time than the rest of the blot to prevent overexposure. C is DNA
from a
non-transformed control M. truncatula plant.
Fig. 2(b) is a Southern blot of Hind III digested DNA from transgenic plants
(progeny of infiltrated plants) hybridized with a bar probe. Samples 1.3-1.8
are from
Treatment 1 (1-minute vacuum infltration); Samples 2.2-2.7 are from Treatment
2
(1.5-minute vacuum infiltration); and Samples 3.1-3.5 are from Treatment 3 (40-
second
CA 02352488 2001-05-25
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vacuum infiltration followed by 20-second hold). The pBI121-bar plasmid DNA is
included to the left. This part of the blot was excised and exposed for a
shorter time
than the rest of the blot to prevent overexposure. C is DNA from a non-
transformed
control M. truncatula plant.
Fig. 3(a) is a Southern blot of HindIII digested DNA from transgenic plants
(progeny of infiltrated plants) hybridized with a npt II probe. Samples 1.6-
1.20 are
from Treatment l (1-minute vacuum infiltration) and Samples 2.12 and 2.13 are
from
Treatment 2 (l.l S-minute vacuum infiltration). C is DNA from a non-
transformed
control M. truncatula plant.
Fig. 3(b) is a Southern blot of HindIII digested DNA from iransgenic plants
(progeny of infiltrated plants) hybridized with a bar probe. Sample 1.6-1.20
are from
Treatment 1 ( 1-minute vacuum infiltration); and Sample 2.12 and 2.13 are from
Treatment 2 ( 1.5-minute vacuum infiltration). C is DNA from a non-transformed
control M. truncatula plant.
t5 Fig. 4 is an agarose gel showing a portion of the bar gene that has been
amplified from DNA from transgenic soybean plants via PCR with bar specific
primers. The arrow points to a 423bp amplified fragment. The lane labeled "M"
contains molecular weight markers. The 500 by marker is indicated. Samples 6-
27 are
soybean transformants that survived the herbicide treatment. Transformants 6,
13, 14,
1 S, and 16 show an amplified fragment of the correct size.
SUMMARY OF THE INVENTION
In one aspect, the present invention is a method for direct plant
transformation
using seedlings and Agrobacterium comprising: (a) contacting at least one
seedling
with Agrobacterium cells which harbor a vector that enables the Agrobacterium
cells to
transfer T-DNA containing at least one gene or gene fragment to the seedling
and (b)
applying a vacuum to the seedling in contact with the Agrobacterium cells at
one point
in time, the vacuum being of sufficient strength to force the Agrobacterium
cells into
intimate contact with the seedling such that the Agrobacterium cells transfer
the T-
3o DNA to cells of the seedling at a second point in time, wherein the first
point in time
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WO 00/37663 PCT/US99/30972
and the second point in time are either the same or different. In a preferred
method, the
vector comprises a selectable marker gene. A preferred selectable marker gene
is a
herbicide resistance gene. A preferred herbicide resistance gene is a bar
gene.
In another aspect, the present invention is a method for direct plant
transformation using seedlings and Agrobacterium comprising: (a) contacting at
least
one seedling with a mixture of Agrobacterium cells, the mixture comprising
cells from
a Agrobacterium strain harboring a vector with a first DNA fragment and cells
from the
Agrobacterium strain harboring the vector with a second DNA fragment, wherein
the
vector enables the Agrobacterium cells to transfer the T-DNA to cells of the
seedling;
1 o and (b) applying a vacuum to the seedling in contact with the mixture of
Agrobacterium
cells at a first point in time, the vacuum being of sufficient strength to
force the
Agrobacterium cells into intimate contact with the seedling such that the
Agrobacterium cells transfer at least one gene to cells of the seedling at a
second point
in time, wherein the first point in time and the second point in time are the
same or
15 different. In a preferred method, the vector comprises a selectable marker
gene. A
preferred selectable marker gene is a herbicide resistance gene. A preferred
herbicide
resistance gene is a bar gene.
In another aspect, the present invention is a method for direct plant
transformation using seedlings and Agrobacterium comprising: (a) contacting at
least
2o one seedling with Agrobacterium cells which harbor a vector that enables
the
Agrobacterium cells to transfer T-DNA containing at least one gene or gene
fragment
and a selectable marker gene to the seedling; (b) applying a vacuum to the
seedling in
contact with the mixture ofAgrobacterium cells at a first point in time, the
vacuum
being of sufficient strength to force the Agrobacterium cells into intimate
contact with
25 the seedling such that the Agrobacterium cells transfer the T-DNA to cells
of the
seedling at a second point in time, wherein the first point in time and the
second point
in time are the same or different; (c) allowing the transformed seedling to
grow to
maturity and set seed; (d) germinating the seed to form progeny; (e) exposing
the
progeny to an agent enabling detection of selectable marker gene expression;
and (f)
3o selecting for progeny expressing the selectable marker gene and at least
one gene,
wherein expression of the selectable marker gene and at least one gene
indicates gene
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WO 00/37663 PCT/US99/30972
transfer. In a preferred method, the selectable marker gene is a herbicide
resistance
gene. A preferred herbicide resistance gene is a bar gene.
In yet another aspect, the present invention is a plant transformed according
to
the above-described methods of seedling transformation.
In yet another aspect, the present invention is a seed from a plant
transformed
according to the above-described methods of seedling transformation.
In yet another aspect, the present invention is a progeny plant from a seed
obtained from a plant transformed according to the above-described methods of
seedling transformation.
DETAILED DESCRIPTION
A plant transformation process has now been found which utilizes vacuum
infiltration of seedlings to introduce Agrobacterium T-DNA carrying a
selectable
marker gene and the genes) of interest into the seedlings. A seedling as used
herein is
defined as a plant from about the beginning of seed germination to about the
time true
leaves develop. The transformation methods described herein can be applied to
the
seedlings of any plant, including divots and monocots which can be
successfully
transformed by Agrobacterium-mediated gene transfer. In particular, leguminous
plants are transformed at high rates of efficiency.
2o The transformation method described herein is generally accomplished by
growing the Agrobacterium strain carrying a genes) of interest under selective
conditions in liquid culture until it reaches exponential growth phase. The
Agrobacterium cells are then pelleted by centrifugation and resuspended in a
vacuum
infiltration medium. The seedlings are immersed in the Agrobacterium cell
suspension
and subjected to vacuum infiltration whereby the Agrobacterium cells are then
introduced into the seedlings, resulting in infiltrated plants that
subsequently produce
transformed seed from which a transformed plant is obtained.
The transformation of seedlings is accomplished through Agrobacterium-
mediated gene transfer. Agrobacterium strains useful in the transformation of
a
CA 02352488 2001-05-25
_ WO 00/37663 PCT/US99/30972
seedling include any aggressive strain which, upon contact with a
transformable plant
cell, is capable of transferring T-DNA into the cell for integration into the
plant's
genome. In the transformation method described herein, the Agrobacterium
strain can
carry one plasmid with multiple genes) of interest. Alternatively,
transformation is
performed using a mixture of Agrobacterium cells in which the vector carries
different
fragments of DNA, e.g., selected fragments from a specific DNA library. To
achieve
the optimum transformation rate in a given plant, the Agrohacterium strain
which
provides the greatest number of transformed seedlings is selected. For
leguminous
plants, Agrobacterium tumefaciens EHA105, ASE1, and Gv3101 strains are
preferably
utilized. The genes) of interest can be transformed into the Agrobacterium by
any
means known in the art. For example, a DNA fragment modified to contain the
genes)
of interest can be inserted into the T-DNA of an Agrobacterium Ti plasmid
which also
contains genes required to generate the transformed state.
Other modifications of the Agrobacterium plasmid T-DNA can be made to
15 assist in the transformation process. For example, to distinguish seedlings
which are
successfully transformed, a selectable marker gene can be incorporated into
the T-DNA
of Agrobacterium plasmid. Selectable marker genes useful in the transformation
methods described herein include any selectable marker gene which can be
incorporated into the Agrobacterium T-DNA and upon expression, can distinguish
2o transformed from non-transformed progeny. Exemplary selectable markers
include a
neomycin transferase gene or phosphinothricin acetyl transferase (bar) gene.
For
example, a preferable selection marker is the bar gene encoding
phosphinothricin
acetyl transferase which confers resistance to phosphinothricin-based
herbicides.
Preferably, the selection marker gene and genes) of interest are incorporated
into any
25 vector suitable for use with transforming Agrobacterium strains. For
example, the
binary vector, pBI121 vector (Clontech, Palo Alto, CA) can be modified wherein
a
copy of a phosphinothricin acetyl transferase (bar) gene is inserted, under
the control of
a 35S promoter and octopine synthase 3' sequences, into the HindIII site of
the T-DNA.
The bar gene encodes phosphinothricin acetyl transferase which confers
resistance to
3o phosphinothricin-based herbicides, such as Ignite~ (AgroEvo, Frankfurt,
Germany).
This selectable marker enables easy selection of transformed plants: upon
spraying the
CA 02352488 2001-05-25
WO 00/37663 PCT/US99/30972
plants with phospinothricin (PPT) containing herbicides, only transformed
plants
containing the bar gene survive exposure to the herbicide.
In the seedling transformation process, the starter seeds can be pretreated to
optimize their germination and to prepare the resulting seedlings for
transformation.
Surface-sterilization of the seeds is preferred to remove any interfering
microorganisms
which might infect the germinating seed. Any sterilization means which does
not
deleteriously affect the seeds can be used. Exemplary methods include use of
aqueous
20-30% sodium hypochlorite or 70% ethanol. Preferably, the seeds are
sterilized in a
solution of 30% sodium hypochlorite and 0.1 % Tween 20 for approximately 5
minutes
1 o and then thoroughly rinsed to remove the sterilizing solution. Preferably,
sterile double
distilled or deionized water, or water with reduced oxidizable carbon
following reverse
osmosis, ion exchange and/or activated charcoal treatment is used to rinse the
seeds.
Some seeds, for example M. truncatula, experience a prolonged dormancy period
resulting in delayed germination. These seeds can be treated by a
scarification process
15 capable of breaking the dormancy. For example, cracking or scratching the
seed coat,
soaking the seed to soften the seed coat, or a controlled acid treatment can
be utilized.
Preferably, a treatment in concentrated sulfuric acid for approximately 10
minutes
followed by thorough rinsing to remove the acid is utilized. Preferably,
sterile double
distilled or deionized water, or water with reduced oxidizable carbon
following reverse
20 osmosis, ion exchange and/or activated charcoal treatment is used to rinse
the seeds.
After pretreatment, the seeds are placed on a medium capable of supporting
germination and subsequent growth of the seedlings. For example, the seeds can
be
placed on the surface of sterile filter paper or paper towels. Preferably, the
seeds are
spread on the surface of firm, sterile water agar in petri plates. The seeds
are then
25 placed under environmental conditions capable of inducing germination and
supporting
development of seedlings. Vernalization may be preferred for certain plants
such as M.
truncatula to promote early flowering. Incubation of the resulting seedlings
is
continued until the seedlings reach an appropriate stage of development for
vacuum
infiltration. The optimum age of seedlings for vacuum infiltration varies for
different
3o plants. In general, seedlings in which the radical has emerged and grown to
at least
about 1 cm are sufficiently mature. However, since plants develop at different
rates,
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WO 00/37663 PCT/US99/30972
vacuum infiltration can be optimized for a specific plant by screening
seedlings at
various stages of development using the methods disclosed herein and
determining the
stage at which the transformation efficiency is maximized. The time and
temperature
of incubation can also be adjusted to provide optimum conditions for a
specific variety.
For example, approximately 15 days after the seeds are placed on germination
medium,
M. truncatula and soybean seedlings are sufficiently matured for vacuum
infiltration.
A few days prior to vacuum infiltration, the transforming Agrobacterium is
subcultured on a general plated growth medium preferably containing
appropriate
antibiotics to distinguish transformed Agrobacterium cells. For example,
to Agrobacterium tumefaciens EHA105 and Gv1301 carrying the bar gene are
preferably
cultured on YEP medium as defined in Example 1 containing rifampicin (20mg/I)
and
kanamycin (SO mg/I). The Agrobacterium cultures are grown at about 28°C
for about
2-3 days.
One day prior to the vacuum infiltration, a liquid Agrobacterium culture is
15 prepared by aseptically transfernng an appropriate inoculum into a general
growth
medium suitable for growing Agrobacterium. TY liquid medium and YEP liquid
medium containing appropriate antibiotics to select for the transformed
Agrobacterium
are preferred forAgrobacterium EHA105 and Gv1301. The liquid cultures are
grown
under conditions which provide the Agrobacterium to reach exponential growth.
2o Preferably, the liquid culture is incubated at about 28°C in a
shaker incubator at about
250 rpm overnight. It is essential to use fresh Agrobacterium to achieve
transformation.
To provide optimal conditions for transformation, the vacuum infiltration is
preferably performed using the transforming Agrobacterium liquid culture in
25 exponential growth phase (OD6~ = 1.6). The Agrobacterium cells in the
liquid culture
are pelleted by centrifugation and resuspended in two volumes of a vacuum
infiltration
medium (e.g., Agrobacterium cells grown in 15m1 liquid culture, pelleted by
centrifugation, and then resuspended in 30m1 vacuum infiltration medium). Any
plant
growth medium capable of supporting the infiltration process and the
Agrobacterium
3o within the plant while being compatible with plant growth can be used as
the vacuum
13
CA 02352488 2001-05-25
WO 00/37663 PCT/US99/30972
infiltration medium. More preferably, the vacuum infiltration medium comprises
acetosyringone which induces the vir genes of the Agrobacterium. For
leguminous
plants, the vacuum infiltration medium defined in Examples 1 and 2 is
preferably
utilized.
To perform the vacuum infiltration, the seedlings are removed from the
germination/incubation medium and placed in any clean container capable of
holding
several seedlings as well as a volume of vacuum infiltration medium to
partially cover
the seedlings. Petri plates are useful for this purpose, using about 30-40
seedlings per
plate. The Agrobacterium suspension in the vacuum infiltration medium is added
to the
to container to wet and partially cover the seedlings. For a standard petri
plate,
approximately 1 Oml of the suspension is sufficient. The petri plate
containing the
seedlings in Agrobacterium suspension is placed in a vacuum chamber. The
preferred
amount of vacuum to be used in the transformation process is the minimal
amount
necessary to force the Agrobacterium into the apoplastic spaces of the
seedlings.
15 Approximately 28mmHg was sufficient for transforming M. truncatula and
soybean.
The time and manner in which the vacuum is applied to the seedlings depends
upon the
plant and has to be determined empirically. The vacuum can be applied then
released.
Alternately, the vacuum can be applied, released, reapplied, and then released
again.
The duration of vacuum can vary from about 0.1 to about 5 min, more preferably
from
2o about 0.5 to about 2 min, and most preferably for about 1 min. For M.
truncatula,
plants held under vacuum for 0.5 min and for 2 min gave rise to transgenic
plants, but
plants held under vacuum for 1 min gave the maximum transformation efficiency.
Following vacuum infiltration, the Agrobacterium suspension is decanted, and
the seedlings are blotted on sterile filter paper or blotting paper. The
seedlings can then
25 be planted into a complete soil mix that will allow full growth and
development of the
plant and the production of seed. The seedlings are then permitted to mature
and set
seed. Preferably, the plants are kept at a humidity, temperature, duration of
photo
period, and spectrum of light which favor plant growth. To increase the
viability of the
transformed plants and to improve the transformation efficiency, the seedlings
are
30 optionally incubated on a co-cultivation medium for 2-3 days prior to
planting in a
complete soil mix. Any co-cultivation medium which supports growth of the
seedlings
14
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can be used. For leguminous plants, the co-cultivation medium given in
Examples 1
and 2 is preferred. The plants are then permitted to develop to maturity and
set seed. A
portion of the seeds will carry the transgene in their genomes. The seeds are
germinated, and the resulting progeny which exhibit stable inheritance of the
transgene
are selected.
Several methods known in the art can be used to distinguish the progeny
exhibiting stable inheritance of the transgene. For transgenic plants wherein
the
genes) of interest results in a visible phenotypic change, the selection can
be based
upon visual examination of the progeny. For plant transformations involving
1 o Agrobacterium carrying plasmids containing a selectable marker gene, the
appropriate
selectable agent can be applied to the plants to select the transformants.
Optionally,
Southern blot analysis or PCR analysis can be used to verify the presence of
the
transferred gene in the genome of the transformed plants.
The seedling transformation processes of the present invention are further
1 s illustrated in detail in the examples provided below. While these examples
describe the
invention, it is understood that modifications to the methods to optimize
transformation
of a specific plant are well within the skill of one in the art, and such
modifications are
considered within the scope of the invention.
Exarnnle 1: Transformation of M. truncatula by Vacuum-infiltration of
Seedlings
2o M. truncatula seedlings were transformed to incorporate the bar gene and
the
nptll gene into the plant's genome using the transformation process of the
present
invention.
Preliminary
Prior to transformation, a modified version of the binary vector, pBI121
vector
25 (Clontech, Palo Alto, CA) was made by inserting a copy of a
phosphinothricin acetyl
transferase (bar) gene, under the control of a 35S promoter and octopine
synthase 3'
sequences, into the HindIII site of the T-DNA to create a plasmid called
pBI121-bar
(Fig. 1 ). The construct was confirmed by restriction analysis and PCR
analysis, and
then transformed into an Agrobacterium tumefaciens strain EHA105 (Hood, et al.
1993.
30 "New A,grobacterium helper plasmids for gene transfer to plants," Trans Res
2:208-
CA 02352488 2001-05-25
WO 00/37663 PCT/US99/30972
218). Additional constructs in Agrobacterium were also obtained as given in
Table III.
While the following procedure is presented for the pBI121-bar in the EHA105
Agrobacterium strain, the same procedure was followed for the other constructs
with
the exception that the growth medium was supplemented with specific
antibiotics
necessary for maintaining the plasmid.
Day 1:
The M. truncatula seed was sterilized and germinated as follows. The seeds
were soaked in conc. HzS04 for approximately 10 min. The acid was removed, and
the
seeds were rinsed extensively in sterile cold double distilled water. This
treatment was
1 o used to break dormancy in M. truncatula.
The seeds were then surface-sterilized by soaking the seeds in a sterilizing
solution such as 30% Clorox / 0.1 % Tween 20 solution for approximately 5 min
with
gentle agitation. The seeds were rinsed extensively with sterile cold double
distilled
water.
The seeds were then spread on a firm water-agar (for example, 0.8%) (Sigma
Chemical Co., St. Louis, MO) in petri plates. The water-agar petri plates
containing the
seeds were wrapped with aluminum foil and kept at 4°C fox 15 days. This
vernalization
step was used to promote early flowering of M. truncatula.
About Day 12:
2o Agrobacterium tumefaciens EHA105 carrying pBI121-bar was subcultured for
isolation onto a fresh agar plate containing YEP medium [1 liter: l Og Bacto-
peptone
(Difco, Detroit, MI); lOg yeast extract; Sg NaCI; and 15g Bacto-agar (Difco,
Detroit,
MI) at pH=6.8 without adjusting] containing rifampicin (20 mg/1) and kanamycin
(50
mg/1), and the subculture was incubated at approximately 28°C for about
2-3days.
About Day 14:
One loop, or approximately 3 large colonies of the Agrobacterium subculture
was inoculated into about 15 ml TY liquid medium [1 liter: Sg tryptone, 3g
yeast
extract, 0.88g CaC12~2Hz0 at pH=7] containing rifampicin (20 mg/1) and
kanamycin
(50 mg/1) and incubated on a 28°C shaker at 250 rpm overnight.
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About Day 15:
The Agrobacterium liquid culture was grown until an exponential phase (ODboo
1.6) was reached. The Agrobacterium cells were pelleted by centrifugation and
resuspended in 30 ml of vacuum-infiltration medium (VIM) [1 liter: l Oml PDM
salt
solution at 100X concentration (400m1 at 100X: 100g KN03; 12g NHQHZP04; 16g
MgS04~7H20; 0.4g MnS04~H20; 0.2g H3B03; 0.008g CuS04~5H20; 0.04g KI; 0.004g
CoC12~6Hz0; 0.04g ZnS04~7H20, and 0.004g Na2Mo04~2H20; filter sterilized and
stored at room temperature); l Oml PDM iron and vitamins (1 liter at 100X:
O.Sg
nicotinic acid; O.OSg pyridoxine~HCl; 0.5g thiamine~HCl; 100g myo-inositol;
1.5g
1o FeS04~7H20; and 2g Na2EDTA; filter sterilized; stored for immediate use at
4°C; long
term storage at -20°C); 0.2g CaC12~H20; 1.5m1 of IOmM benzylaminopurine
(BAP; 0.0565g in 0.15m12N NaOH and 24.85m1 double distilled H20 stored at
4°C);
0.05m1 of IOmM alpha-naphthaleneacetic acid (NAA; 0.0465g in 3m195% ethanol
and
22m170% ethanol stored at 4°C); lOg sucrose; and O.ImI acetosyringone
(AS; 1M in
DMSO stored at -20°C ), wherein PDM salts, PDM iron and vitamins,
CaCl2~Hz0 and
sucrose are combined, the pH adjusted to 5.8 with KOH and autoclaved on liquid
cycle
for 20 min, and when the medium cools to 50°C, BAP, NAA, and AS are
added).
The seedlings were removed from the water agar plates and placed in a clean
standard petri dish at approximately at 30-40 M. truncatula seedling per petri
plate.
2o Approximately 1 Oml of the Agrobacterium suspension in the vacuum
infiltration
medium was added to the petri plate, a volume sufficient to wet and partially
cover the
seedlings. The petri plates containing the seedlings wetted with Agrobacterium
suspension were placed in a vacuum chamber. Three methods of vacuum
infiltration
were tested. In Treatment l, a vacuum was drawn to 28mmHg for approximately 1
min, released rapidly, redrawn to 28mmHg for approximately 1 min, and finally
released rapidly. In Treatment 2, a vacuum was drawn to 28mmHg for
approximately
1.5 min, released rapidly, redrawn to 28mmHg for approximately 1.5 min, and
finally
released rapidly. In Treatment 3, a vacuum was drawn to 28mmHg for
approximately
40 seconds, held for 20 seconds, and finally released rapidly. For all
treatments, the
3o seedlings were then blotted on sterile filter paper or blotting paper and
spread onto petri
plates containing co-cultivation medium(CM) [1 liter: l Oml PDM salt solution
at 10X
17
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- WO 00/37663 PCT/US99/30972
concentration; l Oml PDM iron and vitamins; 0.2g CaC12~H20; lOg sucrose; 7.Sg
agar-
agar (Sigma Chemical Co., St. Louis, MO); and 0.1 ml AS, wherein PDM salts,
PDM
iron and vitamins, CaC12~H20, agar-agar, and sucrose are combined, the pH
adjusted to
5.8 with KOH and autoclaved on liquid cycle for 20 min, and when the medium
cools
to 50°C, AS is added]. The seedlings were incubated in a growth chamber
under the
conditions given in Table I for approximately 2-3 days.
Table I: Growth Chamber Conditions
Temp. Humidity photo-period Light
( C) ( /o)
SET 20 90 16 hrs Top light only
(Day & (8am-l2midnight) (1/2 fluorescent, no
Night) incandescent)
4 light bulbs
Sylvania 115W, F48T1
About Day 17:
1o The seedlings were washed twice with HZO and then planted in pots in Metro-
mix 200 soil mixture. To allow the plants to adjust slowly to ambient humidity
the
following procedure was followed: the pots containing the seedlings were
initially
covered with a plastic cover; after one week, the cover was propped open, and
after a
couple of days, the cover was removed completely. The plants were allowed to
mature
under conditions that are suitable for optimal plant growth, i.e., at 22-
25°C with
eighteen hour days.
About Day 40
The plants began to flower at approximately 24 days after planting in soil.
The
resulting seeds were collected and germinated under conditions optimal for
2o germination, i.e., a short cold treatment for four days on a damp filter
paper, left at
room temperature for 1-2 days, and then planted in soil. When the seedlings
had a few
leaves (approximately 15 days old), they were sprayed with 80 mg/L PPT (
1/7000
dilution of 600mg/ml solution stored at -20°C), and the results are
presented below.
1s
CA 02352488 2001-05-25
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In this study, 120 M. truncatula seedlings were vacuum infiltrated with
Agrobacterium tumefaciens strain EHA105 carrying the pBI121-bar plasmid as
described above. Three different treatments were used, and forty seedlings
were
infiltrated per treatment. The treatments varied only in the length of time of
the
vacuum infiltration treatment. The infiltrated plants were permitted to mature
and set
seed. The progeny seedlings were sprayed with Ignite (PPT) and resistant
seedlings
were further analyzed for the presence of the bar, nptll and B-Glucuronidase
(GUS)
genes. The results are shown in Table II . Fig. 2A, 2B, 3A, and 3B present
data
obtained in Experiments T-84-1, T84-2, and T84-3 of Table II. These results
show the
to efficiency of transformation ranging from 2.9% to 27.6% for the various
transformation
experiments.
Table II: Transformants Resulting from Transformation of M. truncatula
via Infiltration of Seedlings with Agrobacterium
Approx. No.
of
Construct No. of No. No. of Seedlings% % indepen-
and of
Experi-Agrobac- Plants Seed Seedlingsresistanttransfor-dent trans-
ment' teriumb InfiltratedCollectedGerminatedto PPT mation formants
(*)
T84-1 pB1121-bar
/EHA 105 40 1326 329 22 6.7 78
T84-2 pBI121-bar
/EHA 105 40 1263 302 16 5.3 ND'
T84-3 pBI121-bar
/EHA 105 40 1214 173 S 2.9 ND
T87-1 pGA482-bar
/EHA 105 40 ND 217 10 4.6 86
T87-2 pGA482-bar
/EHA105 40 ND 502 89 17.7 78
T87-3 pKYLX7IGus _
/EHA105 40 ND ND ** ND ND
T87-4 pBINmgfp-ER-
Gar l EHA 40 ND 382 13 3.4 8
105
T8?-7 pBINmgfp-ER-
bar and
pKYLX7IGus40 ND 210 58 27.6 ND
/ EHA I
OS -
T88 pSKI015/
Gv3101 70 ND 565 40 7.1 ND
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WO 00/37663 PCT/US99/30972
a The following treatment methods were used: T84-l, T87-I, T87-2, T87-3,
T87-4, T87-7, and T88, infiltration for I min (2X); T84-2, infiltration forl.5
min (2X);
T84-3, infiltration for 40 sec and hold 20 sec.
~ The following Agrobacterium tumefaciens strains and binary vectors were
used in these experiments: A. tumefaciens strain ASE1 carrying the binary
vectors
pSLJ525 (Jones et al. 1992. "Effective vectors for transformation, expression
of
heterologous genes, and assaying transposon excision in transgenic plants,"
Trans. Res.
1: 285-297) or pSKI006 (http://www.salk.edu /LABS/pbio-w/). A. tumefaciens
strain
EHA105, carrying pBI121-bar or PKYLX7-Gus (Franklin et al. 1993 "Genetic
1 o transformation of green bean callus via Agrobacterium mediated DNA
transfer," Plant
Cell Rep 12:74-79), or pBINmgfp-ER-bar, or pGA482-bar. A. tumefaciens strain
Gv3 I O1 carrying pSKI015 (Kardailsky et al. 1999. "A pair of related genes
with
antagonistic roles in floral induction," Science 286, 1962-1965). The addition
of the
bar gene to a number of vectors was achieved as follows. A HindIIIlHpaI
fragment
containing the 35S-bar-OCS 3'sequences cassette was excised from pSLJ525 and
inserted between the HindIII and HpaI sites of pGA482 to create pGA482-bar.
The
same HindIIIlHpaI fragment was used to produce pBI121-bar and pBINmgfp-ER-bar.
For each of these vectors, the HpaI site was converted to a HindIII site by
the addition
of an NpaI-HindIII linker and then the HindIII fragment was then inserted into
the
2o HindIII site of pBI121 or pBINmgfp-ER (Haseloff et al. 1997 "Removal of a
cryptic
intron and subcellular localization of green fluorescent protein are required
to mark
transgenic Arabidopsis plants brightly," Proc. Natl. Acad. Sci. USA 94: 2122-
21271997) to create pBI121-bar and pBINmgfp-ER-bar respectively.
ND=not determined.
Transformed plants from Treatments T84-1, T84-2, and T84-3 were also
analyzed by Southern blot analysis to demonstrate the presence of the
transgene within
the plant. DNA was isolated from 22 transgenic plants (12 plants from
Treatment T84-
1, 6 plants from Treatment T84-2, and 4 plants from Treatment T84-3) and
digested
with restriction enzyme HindIII. The digested DNA was separated by
electrophoresis
3o and blotted to nylon membranes. The membranes were probed with an internal
DNA
fragment (450bp) of the bar gene labeled with 32P-dATP. All of the transformed
plants
CA 02352488 2001-05-25
- WO 00/37663 PCTNS99/30972
contained DNA fragments that hybridized to the bar probe indicating that this
gene is
integrated into the genome (Fig. 2A and 2B). The plasmid pBI121-bar DNA was
included on the blot as a positive control, and a sample of DNA from non-
transformed
M. truncatula plant was included as a negative control. As expected the bar
probe
hybridized to the plasmid DNA but not to the DNA from the non-transformed M.
truncatula plant (Fig. 2A and 2B). The expected size of the hybridizing
fragment from
the plasmid is 1.6kb; however, all of the fragments were larger, probably due
to a
rearrangement towards the left border. The blots were stripped and reprobed
with an
internal fragment of the nptll gene (766bp) labeled with 32P-dATP. The nptll
gene is
o carried on the pBI121-bar plasmid, between the right border of the T-DNA and
the bar
gene (Fig. 1). All of the transformed plants contained DNA fragments that
hybridized
to the nptll probe indicating that this gene is also integrated into the
genome. This
combination of digest and probe provided a right border analysis and
demonstrated the
presence of independent transformants. For example, the unique bar-hybridizing
fragments shown with Transformants 1.6, 1.10, 1.11, 1.14, 1.13, and 2.12
provided
evidence that these are independent transformants. Again, the non-transformed
control
plant does not contain DNA capable of hybridizing to this probe (Fig. 3A and
3B). The
pBI121-bar T-DNA also contains a copy of the GUS gene between the bar gene and
the left border {Fig. 1 ); however, this gene could not be detected in the
transformed
2o plants. Loss of genes located between the selectable marker and the left
border have
been previously reported; thus, the lack of the GUS gene in the transformed
plants
confirmed these findings. These results were consistent with the bar Southern
analysis
and offered an explanation of the larger than expected bar hybridizing
fragment. Thus,
it was demonstrated that the GUS gene or any gene of interest must be inserted
in the
plasmid between the bar gene and the right border (at the location of the
nptll gene) to
ensure integration.
Seed from the transgenic plants were collected and germinated, and the
resulting seedlings were sprayed with PPT herbicide. The progeny of the
transgenic
plants were also highly resistant to PPT, indicating that the transgenes are
stable and
3o inherited by the following generation. As shown in Table III, data was
obtained which
21
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- WO 00/37663 PCT/US99/30972
showed that the transgenes were inherited in a stable Mendelian fashion. The
results
show that the lines can be propagated past the T1 generation.
Table III: Segregation Analysis (Phosphinothricin Resistance) of Progeny from
a
Selection of Transformants Prepared by Infiltration of Seedlings
Number of
plants Number of plantsChi-square
Progeny resistant to sensitive to test
from phosphinothricinphosphinothricinagainst
(R) (S)
transformants ratios
(p)a
3R:1 1 SR:1 638:1
S S S
T84-1.14 83 0 -
T84-1.19 44 20 ** _ _
T84-1.20 38 14 ** _ _
T84-2.6 44 21 * * _ _
a * * = p value of >_ 0.05; * = p value of z 0.01; - = p value of 5 0.01.
Example 2: Transformation of Soybean by Vacuum-infiltration of Seedling-s
Soybean seedlings were transformed to incorporate the bar gene into the
plant's
genome using the transformation process of the present invention.
to Day 1:
The seeds were then surface-sterilized by soaking the seeds in 20% sodium
hypochlorite for approximately 5 min with gentle agitation. The seeds were
rinsed for
eight times in sterile double distilled water. The seeds were then placed in a
large
volume of water and allowed to imbibe at room temperature for 3-12 hours.
s The seeds were then spread on a firm water-agar (for example, 0.8%) (Sigma
Chemical Co., St. Louis, MO) in petri plates. The water-agar petri plates
containing the
seeds were wrapped with aluminum foil and kept at 18-20°C for 15 days.
About Day 12:
Agrobacterium tumefaciens Gv3101 which carries the SKI015 vector with a
2o copy of the bar gene was subcultured for isolation onto a fresh agar plate
containing
YEP medium [1 liter: I Og Bacto-peptone (Difco); 1 Og yeast extract; Sg NaCI;
and 1 Sg
Bacto-agar (Difco) at pH=6.8 without adjusting] containing rifampicin (10
mg/1),
22
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WO 00/37663 PCT/US99/30972
kanamycin (SO mg/1), carbenicillin (SO mg/1), and gentamicin (20 mg/1), and
the
subculture was incubated at approximately 28°C for about 2-3days.
About Day 14:
One loop, or approximately 3 large colonies of the Agrobacterium subculture
was inoculated into about 15 ml TY liquid medium [1 liter: Sg tryptone, 3g
yeast
extract, 0.888 CaC12~2Hz0 at pH=7] containing rifampicin {10 mg/1), kanamycin
(50
mg/1), carbenicillin (50 mg/1), and gentamicin (20 mg/1) and incubated on a
28°C
shaker at 250 rpm overnight.
About Day 15:
1 o The Agrobacterium liquid culture was grown until an exponential phase
(OD6oo
1.6) was reached. The Agrobacterium cells were pelleted by centrifugation and
resuspended in 30 ml of vacuum-infiltration medium (VIM) [1 liter: l Oml PDM
salt
solution at lOX concentration; l Oml PDM iron and vitamins; 0.28 CaC12~H20;
1.$ml of
IOmM benzylaminopurine (BAP; 0.05658 in O.15m12N NaOH and 24.85m1 double
15 distilled H20 stored at 4°C); O.OSmI of l OmM alpha-
naphthaleneacetic acid (NAA;
0.04658 in 3ml 95% ethanol and 22m170% ethanol stored at 4°C); l Og
sucrose; and
O.lml acetosyringone (AS; 1M in DMSO stored at -20°C ), wherein PDM
salts, PDM
iion and vitamins, CaC12~H20 and sucrose are combined, the pH adjusted to 5.8
with
KOH and autoclaved on liquid cycle for 20 min, and when the medium cools to
50°C,
20 BAP, NAA, and AS are added].
The seedlings were removed from the water agar plates and placed in a clean
standard petri dish at approximately at 10-20 soybean seedlings per petri
plate.
Approximately 20m1 of the Agrobacterium suspension in the vacuum infiltration
medium was added to the petri plate, a volume sufficient to wet and partially
cover the
25 seedlings. The petri plates containing the seedlings wetted with
Agrobacterium
suspension were placed in a vacuum chamber. A vacuum was drawn to 28mmHg for
approximately 2 min, released rapidly, redrawn to 28mmHg for approximately 2
min,
and finally released rapidly. The seedlings were then blotted on sterile
filter paper or
blotting paper and spread onto petri plates containing co-cultivation
medium(CM) [1
30 liter: lOml PDM salt solution at 10X concentration; lOml PDM iron and
vitamins; 0.28
23
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- WO 00/37663 PCTNS99/30972
CaC12~H20; l Og sucrose; 7.Sg agar-agar (Sigma Chemical Co., St. Louis, MO);
and
O.lml AS, wherein PDM salts, PDM iron and vitamins, CaCI2~H20, agar-agar, and
sucrose are combined, the pH adjusted to 5.8 with KOH and autoclaved on liquid
cycle
for 20 rnin, and when the medium cools to 50°C, AS is added]. The
seedlings were
incubated in a growth chamber under the conditions given in Table IV for
approximately 6-7 days until the Agrobacterium could be seen growing around
the
seedlings on the media.
Table IV: Growth Chamber Conditions
Temp. Humidity photo-period Light
( C) ( /o)
SET 20 90 16 hrs Top light only
(Day & (8am-l2midnight) (1/2 fluorescent, no
Night) incandescent)
4 light bulbs
Sylvania 115W, F48T1
1o About Day 17:
The seedlings were washed twice with HZO and then planted in pots in Metro-
mix 200 soil mixture. To allow the plants to adjust slowly to ambient humidity
the
following procedure was followed: the pots containing the seedlings were
initially
covered with a plastic cover; after one week, the cover was propped open, and
after a
couple of days, the cover was removed completely. The plants were allowed to
mature
under conditions for optimal growth in a greenhouse.
About Da~40
The plants began to flower, and the resulting seeds were collected and
germinated by soaking in water for three hours followed by immediate planting
in soil.
2o When the seedlings had one leaf, they were sprayed with 100 ml/1 PPT (
1/6000
dilution of 600mg/ml solution in 0.1 % Tween 20 stored at -20°C), and
the results are
presented below.
In this study, 30 soybean seedlings were vacuum infiltrated with Agrobacterium
tumefaciens strain Gv3101 carrying the SKI015-bar plasmid as described above.
Fourteen of the thirty infiltrated soybean seedlings survived, were
transplanted into
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WO 00/37663 PC'T/US99/30972
pots, and allowed to mature and set seed. Approximately 700 progeny seeds were
collected, germinated and grown in vermiculite. After the seedlings developed
at least
one true leaf, they were sprayed with PPT herbicide. PCR analysis was
performed to
confirm that the plants which survived the PPT herbicide carried the bar gene
in their
genomes. DNA was extracted from each surviving plant and used as a template in
a
PCR reaction with bar specific primers. A fragment of the expected size was
amplified
from the DNA samples from at least eleven of the transformed plants,
indicating that
these plants had been transformed. The frequency of transformation was around
1.57%.
1o In summary, the seedling transformation process described herein is more
efficient and less labor intensive than previously reported methods. In
addition,
somatic alterations are avoided, and direct introduction of genetic material
into elite
lines is made possible. Large numbers of transgenic plants representing
diverse
integration events can be generated very rapidly and efficiently, and the
transgenes are
t 5 stable and inherited by the subsequent generation. The major difficulty
with
regeneration of Agrobacterium transformed cells through tissue culture is
avoided in
the transformation procedures of the present invention, making it useful for
legumes
such as, soybean, bean and peas for which subsequent regeneration of
Agrobacterium
transformed cells is problematic.
