U.S. patent number RE44,971 [Application Number 13/855,631] was granted by the patent office on 2014-06-24 for method for selection of transformed cells.
This patent grant is currently assigned to Monsanto Technology LLC. The grantee listed for this patent is Monsanto Technology LLC. Invention is credited to Ronald J. Brinker, Paul C. C. Feng, Yuenchun Wan.
United States Patent |
RE44,971 |
Wan , et al. |
June 24, 2014 |
**Please see images for:
( Certificate of Correction ) ** |
Method for selection of transformed cells
Abstract
The invention provides methods for the selection of transgenic
cells. The invention relates to the unexpected finding that cells
expressing a gene conferring tolerance to auxin-like herbicides
such as dicamba may be directly selected from non-transgenic cells
using auxin-like herbicides as a selective agent. In this manner,
plants exhibiting tolerance to auxin-like herbicides can be
directly produced without the need for separate selectable
markers.
Inventors: |
Wan; Yuenchun (Madison, WI),
Brinker; Ronald J. (Ellisville, MO), Feng; Paul C. C.
(Wildwood, MO) |
Applicant: |
Name |
City |
State |
Country |
Type |
Monsanto Technology LLC |
St. Louis |
MO |
US |
|
|
Assignee: |
Monsanto Technology LLC (St.
Louis, MO)
|
Family
ID: |
38656527 |
Appl.
No.: |
13/855,631 |
Filed: |
April 2, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
60811190 |
Jun 6, 2006 |
|
|
|
Reissue of: |
11758656 |
Jun 5, 2007 |
7851670 |
Dec 14, 2010 |
|
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Current U.S.
Class: |
800/278; 800/300;
800/298; 435/415; 800/312; 536/23.7; 536/23.2 |
Current CPC
Class: |
C12N
15/8209 (20130101); C12N 15/8274 (20130101) |
Current International
Class: |
C12N
15/82 (20060101); C07H 21/04 (20060101); A01H
5/00 (20060101); C12N 5/04 (20060101) |
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WO |
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4:1590-1597, 1990. cited by applicant .
Cork and Krueger, "Microbial Tranformations of Herbicides and
Pesticides," Adv. Appl. Microbiology, 36:1-67, 1991. cited by
applicant .
De Block et al., "Engineering herbicide resistance in plants by
expression of a detoxifying enzyme," EMBO J., 6:2513-2518, 1987.
cited by applicant .
Krueger et al., "Isolation and identification of microorganisms for
the degradation of dicamba," J. Agric. Food Chem., 37:534, 1989.
cited by applicant .
Gurbiel et al., "Active site structure of Rieske-type proteins:
electron nuclear double resonance of studies of isotopically
labeled phthalate dioxygenase from Pseudomonas cepacia and Rieske
protein from rhodobacter capsulatus and molecular modeling studies
of a Rieske center," Biochemistry, 35(24):7834-7845, 1996
(Abstract). cited by applicant .
Krueger et al., "Use of dicamba-degrading microorganisms to protect
dicamba susceptible plant species," J. of Agri. and Food Chem.,
39(5):1000-1003, 1991. cited by applicant.
|
Primary Examiner: O Hara; Eileen B
Attorney, Agent or Firm: Dentons US LLP Sisson; Pamela
Parent Case Text
This application claims the priority of U.S. Provisional Patent
Application 60/811,190, filed Jun. 6, 2006, the disclosure of which
is incorporated herein by reference in its entirety.
Claims
What is claimed is:
1. A method for selecting a transformed soybean plant cell
comprising the steps of: a) contacting a population of plant cells
comprising a transgenic soybean plant cell transformed with a
polynucleotide encoding dicamba monooxygenase with medium
comprising .[.auxin-like herbicide.]. .Iadd.dicamba .Iaddend.in an
amount that inhibits the growth of cells from the population
lacking the polynucleotide, wherein the polynucleotide comprises a
nucleic acid sequence selected from: (1) a nucleic acid sequence
encoding the polypeptide of SEQ ID NO: 6, (2) a nucleic acid
sequence comprising the sequence of SEQ ID NO: 5, and (3) a nucleic
acid sequence encoding a polypeptide with at least 90% sequence
identity to the polypeptide of SEQ ID NO:6, wherein the polypeptide
has dicamba monooxygenase activity; and b) selecting the
transformed soybean plant cell from the population of plant cells
based on tolerance exhibited by the transformed cell to the
.[.auxin-like.]. .Iadd.dicamba .Iaddend.herbicide further wherein:
said polynucleotide encoding dicamba monooxygenase is operatively
linked to a chloroplast transit peptide coding sequence.
2. The method of claim 1, comprising culturing the population of
plant cells on a medium lacking the .[.auxin-like herbicide.].
.Iadd.dicamba .Iaddend.prior to step a) and/or between step a) and
step b).
3. The method of claim 2, wherein the medium lacking the
.[.auxin-like herbicide.]. .Iadd.dicamba .Iaddend.contains a
cytokinin.
4. The method of claim 2, wherein the medium lacking the
.[.auxin-like herbicide.]. .Iadd.dicamba .Iaddend.contains 6-benzyl
amino purine (BAP).
5. The method of claim 4, wherein the 6-benzyl amino purine is in a
concentration of about 10 mg/l of medium or less.
6. The method of claim 1, wherein the polynucleotide encoding
dicamba monooxygenase is not genetically linked to a selectable or
screenable marker gene other than dicamba monooxygenase.
7. The method of claim 1, further comprising the step of: c)
regenerating a fertile transgenic soybean plant from the
transformed soybean plant cell.
.[.8. The method of claim 1, wherein the medium contains at least
two auxin-like herbicides..].
.[.9. The method of claim 8, wherein the medium contains dicamba
and 2,4-dichlorophenoxyacetic acid..].
10. The method of claim 1, wherein the population of cells
comprises a cotyledon explant.
11. The method of claim 10, wherein the transformed plant cell is
prepared by Agrobacterium-mediated transformation.
.[.12. The method of claim 1, wherein said auxin-like herbicide
comprises 2,4-D, MCPA or 2,4-DB..].
13. The method of claim 1, wherein said .[.auxin-like herbicide.].
.Iadd.dicamba .Iaddend.comprises dicamba in a concentration of from
about 0.001 mg/L to about 0.02 mg/L.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates generally to the field of plant
biotechnology. More specifically, the invention relates to methods
for selecting transformed plant cells using auxin-like herbicides
as a selective agent.
2. Description of the Related Art
Transgenic crops are currently grown on more than 80.0 million
hectares world-wide. Improved traits provided by transgenes have
significantly increased productivity and in many instances
decreased reliance on herbicides and insecticides that can
potentially contaminate the environment. However, for transgenic
crops to continue to be competitive in the market place, new
value-added traits will be required.
In the production of transgenic plants, a particularly important
step is the selection of transgenic cells. This is because only a
small percentage of cells are typically transformed in any given
transformation protocol. The use of a selectable marker gene allows
those cells containing a marker gene to be selected away from those
that do not. In attempts to stack multiple transgenes in a single
plant, this can become particularly difficult, as multiple
selectable marker genes are required. Additionally, while a number
of selectable markers have previously been described, many do not
confer a trait of any practical agronomic value and thus needlessly
complicate regulatory approval. Alternatively, labor intensive
steps must be taken to attempt to breed selectable markers out of a
given transgenic plant. A selectable marker gene with dual
functions of a selectable marker and a trait would thus be
especially valuable.
Commonly used selectable marker genes for plant transformation are
neomycin phosphotransferase II, isolated from Tn5 and conferring
resistance to kanamycin (Fraley et al., 1983) and hygromycin
phosphotransferase, which confers resistance to the antibiotic
hygromycin (Vanden Elzen et al., 1985). Additional selectable
marker genes of bacterial origin that confer resistance to
antibiotics include gentamycin acetyl transferase, streptomycin
phosphotransferase, aminoglycoside-3'-adenyl transferase, and the
bleomycin resistance determinant (Hayford et al., 1988; Jones et
al., 1987; Svab et al., 1990; Hille et al., 1986).
Other selectable marker genes for plant transformation not of
bacterial origin are available. These genes include, for example,
mouse dihydrofolate reductase, plant
5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactate
synthase (Eichholtz et al., 1987; Shah et al., 1986; Charest et
al., 1990). Among some herbicides that selectable marker genes
confer resistance to are glyphosate, glufosinate, or bromoxynil
(Comai et al., 1985; Gordon-Kamm et al., 1990; Stalker et al.,
1988).
Genes encoding enzymes which inactivate herbicides and other
xenophobic compounds have previously been isolated from a variety
of prokaryotic and eukaryotic organisms. In some cases, these genes
have been genetically engineered for successful expression in
plants. Through this approach, plants have been developed which are
tolerant to the herbicides 2,4-dichlorophenoxyacetic acid (Streber
and Willmitzer, 1989), bromoxynil (Stalker et al., 1988),
glyphosate (Comai et al., 1985) and phosphinothricin (De Block et
al., 1987). While these plants have proven valuable in a commercial
setting, plants tolerant to other herbicides are needed to avoid
over reliance on any single herbicide and to increase options for
managing difficult to control weed species.
In addition to the foregoing herbicides, there are auxin-like
herbicides that mimic or act like natural plant growth regulators
called auxins. Auxin-like herbicides appear to affect cell wall
plasticity and nucleic acid metabolism, which can lead to
uncontrolled cell division and growth. The injury symptoms caused
by auxin-like herbicides includes epinastic bending and twisting of
stems and petioles, leaf cupping and curling, and abnormal leaf
shape and venation.
Dicamba is one example of an auxin-like herbicide and is used as a
pre-emergent and post-emergent herbicide for the control of annual
and perennial broadleaf weeds and several grassy weeds in corn,
sorghum, small grains, pasture, hay, rangeland, sugarcane,
asparagus, turf, and grass seed crops (Crop Protection Reference,
1995). Unfortunately, dicamba can injure many commercial crops and
dicot plants such as soybeans, cotton, peas, potatoes, sunflowers,
and canola are particularly sensitive to the herbicide. Despite
this, auxin-like herbicides are very effective in controlling weed
growth and thus are an important tool in agriculture. This is
underscored by the development of weeds tolerant to other
herbicides.
Recently, a gene for dicamba monooxygenase (DMO) was isolated from
Pseudomonas maltophilia that confers tolerance to dicamba (US
Patent Appln. 20030135879). DMO is involved in conversion of
herbicidal dicamba (3,6-dichloro-o-anisic acid) to a non-toxic
3,6-dichlorosalicylic acid. This gene provides tolerance to dicamba
in plants expressing the DMO gene. However, transformants
containing the gene had to date only been selected using a separate
selectable marker gene and techniques enabling use of a DMO gene as
a direct selectable marker were not described. The need to use a
separate selectable marker complicates the production of plants
tolerant to auxin-like herbicides by requiring an additional gene
on transformation vectors used and also presents regulatory
hurdles.
Thus, there is a need in the art for new selectable marker genes
and new herbicide tolerance genes. Particularly needed is a method
for the selection of cells expressing a gene conferring tolerance
to dicamba and other auxin-like herbicides that can be directly
selected. A selectable marker gene with the dual function of a
marker and a trait would eliminate the costs associated with
preparing and tracking of two expression units during the
development of a product and would facilitate the production of
plants having valuable new traits.
SUMMARY OF THE INVENTION
In one aspect, the invention provides a method for selecting a
transformed plant cell comprising the steps of: a) contacting a
population of plant cells comprising a transgenic plant cell
transformed with a polynucleotide encoding dicamba monooxygenase
with medium comprising auxin-like herbicide in an amount that
inhibits the growth of cells from the population lacking the
polynucleotide, wherein the polynucleotide comprises a nucleic acid
sequence selected from: (1) a nucleic acid sequence encoding the
polypeptide of SEQ ID NO:8, (2) a nucleic acid sequence comprising
the sequence of SEQ ID NO:7, (3) a nucleic acid sequence that
hybridizes to a complement of the nucleic acid sequence of SEQ ID
NO:7 under conditions of 5.times.SSC, 50% formamide and 42.degree.
C., (4) a nucleic acid sequence having at least 70% sequence
identity to the nucleic acid sequence of SEQ ID NO:7, and (5) a
nucleic acid sequence encoding a polypeptide having at least 70%
sequence identity to the polypeptide sequence of SEQ ID NO:8; and
b) selecting the transformed plant cell from the population of
plant cells based on tolerance exhibited by the transformed cell to
the auxin-like herbicide. The population of cells may be contacted
with medium comprising auxin-like herbicide any amount of time that
allows selection of the transgenic cell. In certain embodiments,
this may comprise at least 1-3 hours or may carried out
indefinitely, for example, for tens or even hundreds of days. In
one embodiment, the method may comprise culturing the population of
plant cells on a medium lacking the auxin-like herbicide prior to
step a) and/or between step a) and step b). The medium lacking the
auxin-like herbicide may contain a cytokinin such as 6-benzyl amino
purine (BAP). In particular embodiments, 6-benzyl amino purine may
be in a concentration of about 10 mg/l of medium or less, including
about 8, 6, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, and about 0.5 mg/l
or less.
In certain embodiments of the invention, a polynucleotide encoding
dicamba monooxygenase is not genetically linked to a selectable or
screenable marker gene other than dicamba monooxygenase. The
polynucleotide encoding dicamba monooxygenase may be operatively
linked to a chloroplast transit peptide. A method of the invention
may also further comprise the step of: regenerating a transgenic
plant from the transformed plant cell. In certain aspects of the
invention, the transformed plant cell is from a dicot or monocot
plant. Examples of dicot plants include alfalfa, beans, broccoli,
cabbage, carrot, cauliflower, cotton, pea, rapeseed, and soybean
and monocots include corn, onion, rice, sorghum, and wheat. In
specific embodiments, the plant is a cotton, soybean or canola
plant.
In certain aspects, an auxin-like herbicide is selected from the
group consisting of a phenoxy carboxylic acid compound, a benzoic
acid compound, a pyridine carboxylic acid compound, a quinoline
carboxylic acid compound, and a benazolinethyl compound. In one
embodiment, a phenoxy carboxylic acid compound is selected from the
group consisting of 2,4-dichlorophenoxyacetic acid,
4-(2,4-dichlorophenoxy) butyric acid, and
(4-chloro-2-methylphenoxy)acetic acid. In specific embodiments, a
2,4-dichlorophenoxyacetic compound, 4-(2,4-dichlorophenoxy) butyric
acid, and/or (4-chloro-2-methylphenoxy)acetic acid is contained in
the medium at a concentration of from about 0.001 mg/l to about 10
mg/l, including, for example, from about 0.01 mg/l to about 10
mg/l, from about 0.01 mg/l to about 5 mg/l, from about 0.1 mg/l to
about 5 mg/l, from about 1 mg/l to about 5 mg/l, from about 1 mg/l
to about 10 mg/l, from about 5 mg/l to about 10 mg/l, and from
about 0.1 mg/l to about 3 mg/l. In other embodiments the benzoic
acid is dicamba (3,6-dichloro-o-anisic acid) and is contained in
the medium at a concentration of from about 0.001 mg/l to about 10
mg/l, including, for example, from about 0.01 mg/l to about 10
mg/l, from about 0.01 mg/l to about 3 mg/l, from about 0.001 mg/l
to about 0.1 mg/l, from about 1 mg/l to about 10 mg/l, from about 2
mg/l to about 10 mg/l, and from about 0.001 mg/l to about 1 mg/l.
In particular embodiments, the medium contains at least two
auxin-like herbicides, for example, dicamba and
2,4-dichlorophenoxyacetic acid. In a method of the invention the
population of cells may comprise a cotyledon explant and the
transformed plant cell may be prepared by Agrobacterium-mediated
transformation.
In another aspect, the invention provides a transgenic plant cell
comprising a polynucleotide encoding dicamba monooxygenase and
capable of growing in medium comprising 0.01 mg/l dicamba, wherein
the dicamba monooxygenase is not genetically linked to a selectable
or screenable marker gene and wherein the polynucleotide encoding
dicamba monooxygenase comprises a nucleic acid sequence selected
from the group consisting of (1) a nucleic acid sequence encoding
the polypeptide of SEQ ID NO:8, (2) a nucleic acid sequence
comprising the sequence of SEQ ID NO:7, (3) a nucleic acid sequence
that hybridizes to a complement of the nucleic acid sequence of SEQ
ID NO:7 under conditions of 5.times.SSC, 50% formamide and
42.degree. C., (4) a nucleic acid sequence having at least 70%
sequence identity to the nucleic acid sequence of SEQ ID NO:7, and
(5) a nucleic acid sequence encoding a polypeptide having at least
70% sequence identity to the polypeptide sequence of SEQ ID NO:8.
The cell may be defined in particular embodiments as prepared by a
selection method disclosed herein The invention also provides a
tissue culture comprising such a cell. The tissue culture may
comprise the cell in a media comprising auxin-like herbicide in an
amount that inhibits the growth of a plant cell of the same
genotype as the transgenic plant cell that lacks the
polynucleotide. The invention still further provides a transgenic
plant regenerated from the transgenic plant cell.
BRIEF DESCRIPTION OF THE FIGURES
The following drawings form part of the present specification and
are included to further demonstrate certain aspects of the present
invention. The invention may be better understood by reference to
one or more of these drawings in combination with the detailed
description of specific embodiments presented herein.
FIG. 1. Response of soybean explants to dicamba with or without
addition of BAP. (A) On medium without dicamba (left) or with 0.1
mg/l dicamba (right), 13DAT. (B) Explants were inoculated and
co-cultivated with Agrobacterium for 3 days, and then cultured on
medium with 0 (top left), 0.1 (top center), 0.5 (top right), 1.0
(bottom left), 5.0 (bottom center) and 10 (bottom right) mg/l
dicamba, 11DAT (14DAI). (C) Explants were also inoculated and
co-cultivated with Agrobacterium for 3 d, and then cultured on
medium with different levels of dicamba combined with BAP. From
left to right: 0, 0.1, 1.0, and 5.0 mg/l dicamba. From top to
bottom: 0, 1.0, 3.0, 5.0 mg/l BAP.
FIG. 2. Examples of explants with GFP+small bud (top) or sectors
(bottom) in experiment (Exp508) with dicamba selection. The
pictures were taken at 45 DAI under regular bright field (left) or
UV light for detecting GFP expression (right).
FIG. 3. GFP-positive event from dicamba selection. (A) A small
shoot observed 29 DAI under regular dissecting microscope. (B) The
same bud showed GFP-expression as observed under fluorescent light
for detecting GFP. (C) The small shoot in A&B developed into a
resistant elongated shoot (arrow), 48DAI.
FIG. 4. Response of explants cultured on medium containing 0.01
(left), 0.02 (center) and 0.05 mg/l dicamba, 23DAT (29 DAI).
FIG. 5. Detached resistant shoots were cultured on the liquid
rooting medium with small glass beads (A) as support material and
almost all of the shoots could produce roots. (B) Semi-solid medium
can also be used for root induction.
FIG. 6. (A) Young soybean flowers from a transgenic plant with CP4
and GUS gene (top) and a plant transformed with pMON73691 through
dicamba selection and also carrying a GFP gene (bottom). (B) The
same two flowers observed under a dissecting microscope equipped
with fluorescent light to detect GFP expression. GFP expression was
observed on the flower transformed with pMON73691, which contains
DMO and GFP genes. (C, D) The same flower transformed with
pMON73691 was opened to show GFP expression in various floral
structures.
FIG. 7. Soybean explants cultured on selection medium containing
0.01 mg/l dicamba after being inoculated with Agrobacterium
harboring different constructs containing different versions of DMO
gene driven with different CTP. (A) pMON73696, DMO-w, with CTP1. (B
and D) pMON73698, DMO-c with CTP1. (C) pMON73691, DMO-c, with CTP2.
The pictures were taken 39 (A&B) and 54 DAI (C&D),
respectively. Resistant shoots are shown by arrow in panels A and
C.
FIG. 8. Shows susceptibility of wild type Arabidopsis to various
concentration of dicamba in culture medium.
FIG. 9. Shows recovery of dicamba tolerant Arabidopsis plants
transformed with a DMO-encoding polynucleotide.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides in one aspect methods for the selection of
transformed cells with auxin-like herbicides such as dicamba. The
invention overcomes deficiencies in the prior art that previously
required coupling of a gene conferring tolerance to auxin-like
herbicides to a separate selectable marker gene in order to recover
transformants. Direct selection eliminates the need for extraneous
selectable marker genes, which can complicate transformation
procedures and subsequent regulatory approval of transgenic plants.
Efficient selection of transgenic cells is crucial because
typically only a small number of cells are transformed in a
transformation protocol. Cells that survive exposure to the
selective agent may then be cultured in media that supports
regeneration of plants to produce transgenic plants. By use of a
nucleic acid encoding dicamba monooxygenase (DMO) in particular,
the invention allows the selection and creation of transgenic
plants exhibiting tolerance to auxin-like herbicides, which can be
applied to fields containing herbicide tolerant plants for
effective weed control.
Selection of transformed cells in accordance with the invention may
be carried out, for example, by first introducing a DMO-encoding
polynucleotide molecule into a selected target plant tissue;
contacting cells containing the transformed plant cell with a
medium containing an auxin-like herbicide in an amount that
inhibits the growth of plant cells of the same genotype as the
transformed plant cell not containing the DMO-encoding
polynucleotide; and selecting a plant cell capable of growing in
the medium. In this manner, a transgenic cell can be selected from
a large population of non-transgenic cells. In an exemplary
embodiment, selective media may be modified by including further
substances such as growth regulators. Tissue may be maintained on a
basic media with growth regulators until sufficient tissue is
available to begin plant regeneration efforts, or following
repeated rounds of manual selection, until the morphology of the
tissue is suitable for regeneration, typically at least 2 weeks,
then transferred to media conducive to maturation into plants.
Cultures may be transferred every 2 weeks on this medium. Shoot
development will signal the time to transfer to medium lacking
growth regulators.
Numerous plant tissues are amenable to transformation. The plant
cell may in certain embodiments come from a plant explant, which
refers to a part excised from a plant that is capable of being
transformed and subsequently regenerated into a transgenic plant.
Typical explants include cell suspensions, meristems, mature or
immature embryos, dry embryos, wet embryos, dried embryos, seeds,
callus, cotyledons, cotyledonary nodes, leaves, or stems.
Once a transgenic cell has been selected and tissues grown
therefrom, the presence of the exogenous DNA or "transgene(s)" in
the regenerating tissue or plants can be confirmed using a variety
of assays. Such assays include, for example, "molecular biological"
assays, such as Southern and northern blotting and PCR.TM.;
"biochemical" assays, such as detecting the presence of a protein
product, e.g., by immunological means (ELISAs and western blots) or
by enzymatic function; plant part assays, such as leaf or root
assays; and also, by analyzing the phenotype of the whole
regenerated plant.
A. Nucleic Acids and Recombinant Constructs
1. Dicamba Monooxygenase (DMO)
In one embodiment of the present invention, a DNA construct
expressing a dicamba monooxygenase (DMO) polypeptide is used as a
selectable marker gene in plant cells. Exemplary DMO polypeptides
are provided herein as SEQ ID Nos: 2, 4, 6, 8, 10 or 12. Exemplary
nucleic acids encoding these sequences are provided as SEQ ID Nos:
1, 3, 5, 7, 9, or 11. Thus, in one embodiment of the invention,
these sequences are used for the selection of transformed cells. As
is well known in the art, homologous sequences and derivatives of
these sequences may readily be prepared and used. For example, a
nucleic acid may be used that encodes a DMO polypeptide having at
least 70% sequence identity to a polypeptides provided as SEQ ID
No: 2, 4, 6, 8, 10 or 12, including at least about 75%, 80%, 85%,
90%, 95%, 97%, 98%, 99% or greater identity to such sequences. A
nucleic acid may be also be used that exhibits at least 70%
sequence identity to a nucleic acid sequence provided as SEQ ID No:
1, 3, 5, 7, 9, or 11, including at least about 75%, 80%, 85%, 90%,
95%, 97%, 98%, 99% or greater identity to such sequences. In one
embodiment, the identity is determined using the Sequence Analysis
software package of the GCG Wisconsin Package (Accelrys, San Diego,
Calif.), MEGAlign (DNAStar, Inc., 1228 S. Park St., Madison, Wis.
53715) with default parameters. Such software matches similar
sequences by assigning degrees of similarity or identity.
A polynucleotide molecule that expresses a DMO polypeptide can be
obtained by techniques well known in the art. Variants of DMOs
having a capability to degrade auxin-like herbicides can readily be
prepared and assayed for activity according to standard methods.
Such sequences can also be identified by techniques know in the
art, for example, from suitable organisms including bacteria that
degrade auxin-like herbicides such as dicamba (U.S. Pat. No.
5,445,962; Krueger et al., 1989; Cork and Krueger, 1991; Cork and
Khalil, 1995). One means of isolating a DMO sequence is by nucleic
acid hybridization, for example, to a library constructed from the
source organism, or by RT-PCR using mRNA from the source organism
and primers based on the disclosed DMO. The invention therefore
encompasses use of nucleic acids hybridizing under stringent
conditions to a DMO encoding sequence described herein. One of
skill in the art understands that conditions may be rendered less
stringent by increasing salt concentration and decreasing
temperature. Thus, hybridization conditions can be readily
manipulated, and thus will generally be a method of choice
depending on the desired results. An example of high stringency
conditions is 5.times.SSC, 50% formamide and 42.degree. C. By
conducting a wash under such conditions, for example, for 10
minutes, those sequences not hybridizing to a particular target
sequence under these conditions can be removed. One embodiment of
the invention thus comprises use of a DMO-encoding nucleic acid
that is defined as hybridizing under wash conditions of
5.times.SSC, 50% formamide and 42.degree. C. for 10 minutes to a
nucleic acid selected from SEQ ID NOS: 1, 3, 5, 7, 9, or 11.
SEQ ID NO: 1 shows DMO from Pseudomonas maltophilia optimized for
expression in dicots using Arabidopsis thaliana codon usage. The
polypeptide, predicted to have an Ala, Thr, Cys at positions 2, 3,
112, respectively, is given in SEQ ID NO:2. SEQ ID NO:3 shows
another Pseudomonas maltophilia DMO optimized for expression in
dicots and encoding the polypeptide of SEQ ID NO:4, predicted to
have an Leu, Thr, Cys at positions 2, 3, 112, respectively. SEQ ID
NO:5 shows the coding sequence and SEQ ID NO:6 the polypeptide for
dicot optimized DMO predicted to have a Leu, Thr, Trp at positions
2, 3, 112, respectively. SEQ ID NOS:7 and 8 show the coding and
polypeptide sequences for DMO predicted to have an Ala, Thr, Cys at
position 2, 3, 112, respectively. SEQ ID NOS:9 and 10 show the
dicot-optimized coding sequence and polypeptide sequences for DMO
predicted to have an Ala, Thr, Trp at positions 2, 3, 112,
respectively. SEQ ID NOS: 11 and 12 show coding sequence and
polypeptide sequences for DMO from Pseudomonas maltophilia (US
Patent Application No: 20030135879).
Variants can also be chemically synthesized using the known DMO
polynucleotide sequences according to techniques well known in the
art. For instance, DNA sequences may be synthesized by
phosphoamidite chemistry in an automated DNA synthesizer. Chemical
synthesis has a number of advantages. In particular, chemical
synthesis is desirable because codons preferred by the host in
which the DNA sequence will be expressed may be used to optimize
expression. Not all of the codons need to be altered to obtain
improved expression, but preferably at least the codons rarely used
in the host are changed to host-preferred codons. High levels of
expression can be obtained by changing greater than about 50%, most
preferably at least about 80%, of the codons to host-preferred
codons. The codon preferences of many host cells are known (PCT WO
97/31115; PCT WO 97/11086; EP 646643; EP 553494; and U.S. Pat. Nos.
5,689,052; 5,567,862; 5,567,600; 5,552,299 and 5,017,692). The
codon preferences of other host cells can be deduced by methods
known in the art. Also, using chemical synthesis, the sequence of
the DNA molecule or its encoded protein can be readily changed to,
for example, optimize expression (for example, eliminate mRNA
secondary structures that interfere with transcription or
translation), add unique restriction sites at convenient points,
and delete protease cleavage sites.
Modification and changes may be made to the polypeptide sequence of
a protein such as the DMO sequences provided herein while retaining
enzymatic activity. The following is a discussion based upon
changing the amino acids of a protein to create an equivalent, or
even an improved, modified polypeptide and corresponding coding
sequences. In particular embodiments of the invention, DMO
sequences may be altered in this manner and used in the methods of
the invention. The amino acid changes may be achieved by changing
the codons of the DNA sequence.
It is known, for example, that certain amino acids may be
substituted for other amino acids in a protein structure without
appreciable loss of interactive binding capacity with structures
such as binding sites on substrate molecules. Since it is the
interactive capacity and nature of a protein that defines that
protein's biological functional activity, certain amino acid
sequence substitutions can be made in a protein sequence, and, of
course, the underlying DNA coding sequence, and nevertheless obtain
a protein with like properties. It is thus contemplated that
various changes may be made in the DMO peptide sequences described
herein and corresponding DNA coding sequences without appreciable
loss of their biological utility or activity.
In making such changes, the hydropathic index of amino acids may be
considered. The importance of the hydropathic amino acid index in
conferring interactive biologic function on a protein is generally
understood in the art (Kyte et al., 1982). It is accepted that the
relative hydropathic character of the amino acid contributes to the
secondary structure of the resultant protein, which in turn defines
the interaction of the protein with other molecules, for example,
enzymes, substrates, receptors, DNA, antibodies, antigens, and the
like. Each amino acid has been assigned a hydropathic index on the
basis of their hydrophobicity and charge characteristics (Kyte et
al., 1982), these are: isoleucine (+4.5); valine (+4.2); leucine
(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine
(+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine
(-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6);
histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate
(-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
It is known in the art that amino acids may be substituted by other
amino acids having a similar hydropathic index or score and still
result in a protein with similar biological activity, i.e., still
obtain a biological functionally equivalent protein. In making such
changes, the substitution of amino acids whose hydropathic indices
are within .+-.2 is preferred, those which are within .+-.1 are
particularly preferred, and those within .+-.0.5 are even more
particularly preferred.
It is also understood in the art that the substitution of like
amino acids can be made effectively on the basis of hydrophilicity.
U.S. Pat. No. 4,554,101 states that the greatest local average
hydrophilicity of a protein, as governed by the hydrophilicity of
its adjacent amino acids, correlates with a biological property of
the protein. As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4). It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent
protein. In such changes, the substitution of amino acids whose
hydrophilicity values are within .+-.2 is preferred, those which
are within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred. Exemplary
substitutions which take these and various of the foregoing
characteristics into consideration are well known to those of skill
in the art and include: arginine and lysine; glutamate and
aspartate; serine and threonine; glutamine and asparagine; and
valine, leucine and isoleucine.
2. Transformation Constructs
A DMO-encoding polynucleotide used in accordance with the invention
as a selectable marker will typically be introduced into a cell as
a construct comprising expression control elements necessary for
the efficient expression of DMO. Methods of operatively linking
expression control elements to coding sequences are well known in
the art (Maniatis et al., 1982; Sambrook et al., 1989). Expression
control sequences are DNA sequences involved in any way in the
control of transcription. Suitable expression control sequences and
methods of using them are well known in the art. A promoter in
particular may be used, with or without enhancer elements, 5'
untranslated region, transit or signal peptides for targeting of a
protein or RNA product to a plant organelle, particularly to a
chloroplast and 3' untranslated regions such as polyadenylation
sites. One skilled in the art will know that various enhancers,
promoters, introns, transit peptides, targeting signal sequences,
and 5' and 3' untranslated regions (UTRs) are useful in the design
of effective plant expression vectors, such as those disclosed, for
example, in U.S. Patent Application Publication 2003/01403641.
Promoters suitable for the current and other uses are well known in
the art. Examples describing such promoters include U.S. Pat. No.
6,437,217 (maize RS81 promoter), U.S. Pat. No. 5,641,876 (rice
actin promoter), U.S. Pat. No. 6,426,446 (maize RS324 promoter),
U.S. Pat. No. 6,429,362 (maize PR-1 promoter), U.S. Pat. No.
6,232,526 (maize A3 promoter), U.S. Pat. No. 6,177,611
(constitutive maize promoters), U.S. Pat. Nos. 5,322,938,
5,352,605, 5,359,142 and 5,530,196 (35S promoter), U.S. Pat. No.
6,433,252 (maize L3 oleosin promoter), U.S. Pat. No. 6,429,357
(rice actin 2 promoter as well as a rice actin 2 intron), U.S. Pat.
No. 5,837,848 (root specific promoter), U.S. Pat. No. 6,294,714
(light inducible promoters), U.S. Pat. No. 6,140,078 (salt
inducible promoters), U.S. Pat. No. 6,252,138 (pathogen inducible
promoters), U.S. Pat. No. 6,175,060 (phosphorus deficiency
inducible promoters), U.S. Pat. No. 6,635,806 (gamma-coixin
promoter), and U.S. patent application Ser. No. 09/757,089 (maize
chloroplast aldolase promoter). Additional promoters that may find
use are a nopaline synthase (NOS) promoter (Ebert et al., 1987),
the octopine synthase (OCS) promoter (which is carried on
tumor-inducing plasmids of Agrobacterium tumefaciens), the
caulimovirus promoters such as the cauliflower mosaic virus (CaMV)
19S promoter (Lawton et al., 1987), the CaMV 35S promoter (Odell et
al., 1985), the figwort mosaic virus 35S-promoter (Walker et al.,
1987), the sucrose synthase promoter (Yang et al., 1990), the R
gene complex promoter (Chandler et al., 1989), and the chlorophyll
a/b binding protein gene promoter, etc. Particularly beneficial
promoters for use with the present invention are CaMV35S, FMV35S,
PClSV, AtAnt1 and P-AGRtu.nos promoters (also see Table 1).
Benefit may be obtained for the expression of heterologous genes by
use of a sequence coding for a transit peptide. Transit peptides
generally refer to peptide molecules that when linked to a protein
of interest directs the protein to a particular tissue, cell,
subcellular location, or cell organelle. Examples include, but are
not limited to, chloroplast transit peptides, nuclear targeting
signals, and vacuolar signals. A chloroplast transit peptide is of
particular utility in the present invention for directing
expression of a DMO enzyme to the chloroplasts. It is anticipated
that DMO function will be facilitated by endogenous reductases and
ferredoxins found in plant cells to degrade dicamba. Plant
chloroplasts are particularly rich in reductases and ferredoxins.
Accordingly, in a preferred embodiment for the production of
transgenic dicamba-tolerant plants a sequence coding for a peptide
may be used that will direct dicamba-degrading oxygenase into
chloroplasts. Alternatively or in addition, heterologous reductase
and/or ferredoxin can also be expressed in a cell.
DNA coding for a chloroplast targeting sequence may preferably be
placed upstream (5') of a sequence coding for DMO, but may also be
placed downstream (3') of the coding sequence, or both upstream and
downstream of the coding sequence. A chloroplast transit peptide
(CTP) in particular can be engineered to be fused to the N-terminus
of proteins that are to be targeted into the plant chloroplast.
Many chloroplast-localized proteins are expressed from nuclear
genes as precursors and are targeted to the chloroplast by a CTP
that is removed during the import steps. Useful CTPs can be
identified from the primary amino acid sequence of such
polypeptides. Examples of chloroplast proteins include the small
subunit (RbcS2) of ribulose-1,5,-bisphosphate carboxylase,
ferredoxin, ferredoxin oxidoreductase, the light-harvesting complex
protein I and protein II, and thioredoxin F. It has been
demonstrated in vivo and in vitro that non-chloroplast proteins may
be targeted to the chloroplast by use of protein fusions with a CTP
and that a CTP is sufficient to target a protein to the
chloroplast. For example, incorporation of a suitable chloroplast
transit peptide, such as, the Arabidopsis thaliana EPSPS CTP (Klee
et al., 1987), and the Petunia hybrida EPSPS CTP (della-Cioppa et
al., 1986) has been shown to target heterologous EPSPS protein
sequences to chloroplasts in transgenic plants. Other exemplary
chloroplast targeting sequences include the maize cab-m7 signal
sequence (Becker et al., 1992; PCT WO 97/41228) and the pea
glutathione reductase signal sequence (Creissen et al., 1991; PCT
WO 97/41228). In the present invention, AtRbcS4 (CTP1), AtShkG
(CTP2), AtShkGZm (CTP2synthetic), and PsRbcS, as well as others,
disclosed in U.S. Provisional Appln. Ser. No. 60/891,675, in
particular may be of benefit, for instance with regard to
expression of a DMO polypeptide (e.g. SEQ ID NOs:17-28 for peptide
sequences of CTPs and nucleic acid sequences that encode them).
A 5' UTR that functions as a translation leader sequence is a DNA
genetic element located between the promoter sequence of a gene and
the coding sequence. The translation leader sequence is present in
the fully processed mRNA upstream of the translation start
sequence. The translation leader sequence may affect processing of
the primary transcript to mRNA, mRNA stability or translation
efficiency. Examples of translation leader sequences include maize
and petunia heat shock protein leaders (U.S. Pat. No. 5,362,865),
plant virus coat protein leaders, plant rubisco leaders, among
others (Turner and Foster, 1995). In the present invention, 5' UTRs
that may in particular find benefit are GmHsp, PhD-naK, AtAnt1,
TEV, and L-Atnos (also see Table 1).
The 3' non-translated sequence, 3' transcription termination
region, or poly adenylation region means a DNA molecule linked to
and located downstream of a structural polynucleotide molecule and
includes polynucleotides that provide polyadenylation signal and
other regulatory signals capable of affecting transcription, mRNA
processing or gene expression. The polyadenylation signal functions
in plants to cause the addition of polyadenylate nucleotides to the
3' end of the mRNA precursor. The polyadenylation sequence can be
derived from the natural gene, from a variety of plant genes, or
from T-DNA genes. An example of a 3' transcription termination
region is the nopaline synthase 3' region (nos 3'; Fraley et al.,
1983). The use of different 3' nontranslated regions is exemplified
(Ingelbrecht et al., 1989). Polyadenylation molecules from a Pisum
sativum RbcS2 gene (Ps.R-bcS2-E9; Coruzzi et al., 1984) and
T-AGRtu.nos (Rojiyaa et al., 1987, Genbank Accession E01312) in
particular may be of benefit for use with the invention.
A DMO-encoding polynucleotide molecule expression unit can be
linked to a second polynucleotide molecule in an expression unit
containing genetic elements for a screenable/scorable marker or for
a gene conferring a desired trait. Commonly used genes for
screening presumptively transformed cells include
.beta.-glucuronidase (GUS), .beta.-galactosidase, luciferase, and
chloramphenicol acetyltransferase (Jefferson, 1987; Teeri et al.,
1989; Koncz et al., 1987; De Block et al., 1984), green fluorescent
protein (GFP) (Chalfie et al., 1994; Haseloff et al., 1995; and PCT
application WO 97/41228). An AvGFP interrupted by StLS1 was used in
the working examples for obtaining expression only in plant cells
(also see Table 1).
The second polynucleotide molecule includes, but is not limited to,
a gene that provides a desirable characteristic associated with
plant morphology, physiology, growth and development, yield,
nutritional enhancement, disease or pest resistance, or
environmental or chemical tolerance and may include genetic
elements comprising herbicide resistance (U.S. Pat. Nos. 6,803,501;
6,448,476; 6,248,876; 6,225,114; 6,107,549; 5,866,775; 5,804,425;
5,633,435; 5,463,175), increased yield (U.S. Pat. RE38,446; U.S.
Pat. No. 6,716,474; U.S. Pat. No. 6,663,906; U.S. Pat. No.
6,476,295; U.S. Pat. No. 6,441,277; U.S. Pat. No. 6,423,828; U.S.
Pat. No. 6,399,330; U.S. Pat. No. 6,372,211; U.S. Pat. No.
6,235,971; U.S. Pat. No. 6,222,098; U.S. Pat. No. 5,716,837),
insect control (U.S. Pat. Nos. 6,809,078; 6,713,063; 6,686,452;
6,657,046; 6,645,497; 6,642,030; 6,639,054; 6,620,988; 6,593,293;
6,555,655; 6,538,109; 6,537,756; 6,521,442; 6,501,009; 6,468,523;
6,326,351; 6,313,378; 6,284,949; 6,281,016; 6,248,536; 6,242,241;
6,221,649; 6,177,615; 6,156,573; 6,153,814; 6,110,464; 6,093,695;
6,063,756; 6,063,597; 6,023,013; 5,959,091; 5,942,664; 5,942,658,
5,880,275; 5,763,245; 5,763,241), fungal disease resistance (U.S.
Pat. Nos. 6,653,280; 6,573,361; 6,506,962; 6,316,407; 6,215,048;
5,516,671; 5,773,696; 6,121,436; 6,316,407; 6,506,962), virus
resistance (U.S. Pat. Nos. 6,617,496; 6,608,241; 6,015,940;
6,013,864; 5,850,023; 5,304,730), nematode resistance (U.S. Pat.
No. 6,228,992), bacterial disease resistance (U.S. Pat. No.
5,516,671), plant growth and development (U.S. Pat. Nos. 6,723,897;
6,518,488), starch production (U.S. Pat. Nos. 6,538,181; 6,538,179;
6,538,178; 5,750,876; 6,476,295), modified oils production (U.S.
Pat. Nos. 6,444,876; 6,426,447; 6,380,462), high oil production
(U.S. Pat. Nos. 6,495,739; 5,608,149; 6,483,008; 6,476,295),
modified fatty acid content (U.S. Pat. Nos. 6,828,475; 6,822,141;
6,770,465; 6,706,950; 6,660,849; 6,596,538; 6,589,767; 6,537,750;
6,489,461; 6,459,018), high protein production (U.S. Pat. No.
6,380,466), fruit ripening (U.S. Pat. No. 5,512,466), enhanced
animal and human nutrition (U.S. Pat. Nos. 6,723,837; 6,653,530;
6,5412,59; 5,985,605; 6,171,640), biopolymers (U.S. Pat. RE37,543;
U.S. Pat. No. 6,228,623; U.S. Pat. No. 5,958,745 and U.S. Patent
Publication No. US20030028917), environmental stress resistance
(U.S. Pat. No. 6,072,103), pharmaceutical peptides and secretable
peptides (U.S. Pat. Nos. 6,812,379; 6,774,283; 6,140,075;
6,080,560), improved processing traits (U.S. Pat. No. 6,476,295),
improved digestibility (U.S. Pat. No. 6,531,648) low raffinose
(U.S. Pat. No. 6,166,292), industrial enzyme production (U.S. Pat.
No. 5,543,576), improved flavor (U.S. Pat. No. 6,011,199), nitrogen
fixation (U.S. Pat. No. 5,229,114), hybrid seed production (U.S.
Pat. No. 5,689,041), fiber production (U.S. Pat. Nos. 6,576,818;
6,271,443; 5,981,834; 5,869,720) and biofuel production (U.S. Pat.
No. 5,998,700). Any of these or other genetic elements, methods,
and transgenes may be used with the invention as will be
appreciated by those of skill in the art in view of the instant
disclosure.
Alternatively, the second polynucleotide molecule can affect the
above mentioned plant characteristic or phenotype by encoding a RNA
molecule that causes the targeted inhibition of expression of an
endogenous gene, for example, via antisense, inhibitory RNA (RNAi),
or cosuppression-mediated mechanisms. The RNA could also be a
catalytic RNA molecule (i.e., a ribozyme) engineered to cleave a
desired endogenous mRNA product. Thus, any polynucleotide molecule
that encodes a transcribed RNA molecule that affects a phenotype or
morphology change of interest may be useful for the practice of the
present invention.
Expression units may be provided on T-DNAs between right border
(RB) and left border (LB) regions of a first plasmid together with
a second plasmid carrying T-DNA transfer and integration functions
in Agrobacterium. The constructs may also contain plasmid backbone
DNA segments that provide replication function and antibiotic
selection in bacterial cells, for example, an Escherichia coli
origin of replication such as ori322, a broad host range origin of
replication such as oriV or oriRi, and a coding region for a
selectable marker such as Spec/Strp that encodes for Tn7
aminoglycoside adenyltransferase (aadA) conferring resistance to
spectinomycin or streptomycin, or a gentamicin (Gm, Gent)
selectable marker gene. For plant transformation, the host
bacterial strain is often Agrobacterium tumefaciens ABI, C58, or
LBA4404. However, other strains known to those skilled in the art
of plant transformation can function in the present invention.
3. Preparation of Transgenic Cells
Transforming plant cells can be achieved by any of the techniques
known in the art for introduction of transgenes into cells (see,
for example, Miki et al., 1993). Examples of such methods are
believed to include virtually any method by which DNA can be
introduced into a cell. Methods that have been described include
electroporation as illustrated in U.S. Pat. No. 5,384,253;
microprojectile bombardment as illustrated in U.S. Pat. Nos.
5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861; and
6,403,865; Agrobacterium-mediated transformation as illustrated in
U.S. Pat. Nos. 5,635,055; 5,824,877; 5,591,616; 5,981,840; and
6,384,301; and protoplast transformation as illustrated in U.S.
Pat. No. 5,508,184. Through the application of techniques such as
these, the cells of virtually any plant species may be stably
transformed and selected according to the invention and these cells
developed into transgenic plants.
The most widely utilized method for introducing an expression
vector into plants is based on the natural transformation system of
Agrobacterium (for example, Horsch et al., 1985). A. tumefaciens
and A. rhizogenes are plant pathogenic soil bacteria which
genetically transform plant cells. The Ti and Ri plasmids of A.
tumefaciens and A. rhizogenes, respectively, carry genes
responsible for genetic transformation of the plant (for example,
Kado, 1991). Descriptions of Agrobacterium vector systems and
methods for Agrobacterium-mediated gene transfer are provided by
numerous references, including Gruber et al., supra, Miki et al.,
supra, Moloney et al., 1989, and U.S. Pat. Nos. 4,940,838 and
5,464,763. Other bacteria such as Sinorhizobium, Rhizobium, and
Mesorhizobium that interact with plants naturally can be modified
to mediate gene transfer to a number of diverse plants. These
plant-associated symbiotic bacteria can be made competent for gene
transfer by acquisition of both a disarmed Ti plasmid and a
suitable binary vector (e.g. Broothaerts et al, 2005; U.S. patent
application Ser. No. 11/749,583).
B. Tissue Cultures and Media
In accordance with the invention transgenic cells may be selected
by using media containing an amount of an auxin-like herbicide that
inhibits the growth of a cell lacking expression of a DMO
polypeptide. "Media" refers to the numerous nutrient mixtures that
are used to grow cells in vitro, that is, outside of the intact
living organism. The medium is usually a suspension of various
categories of ingredients (salts, amino acids, growth regulators,
sugars, buffers) that are required for growth of most cell types.
However, each specific cell type requires a specific range of
ingredient proportions for growth, and an even more specific range
of formulas for optimum growth. Rate of cell growth will also vary
among cultures initiated with the array of media that permit growth
of that cell type.
Regenerating a transformed plant cell can be achieved by first
culturing the explant on a shooting medium and subsequently on a
rooting medium. In accordance with the invention these media
generally include an auxin-like herbicide such as dicamba as the
selection agent besides nutrients and growth regulators. A variety
of media and transfer requirements can be implemented and optimized
for each plant system for plant transformation and recovery of
transgenic plants. Consequently, such media and culture conditions
disclosed in the present invention can be modified or substituted
with nutritionally equivalent components, or similar processes for
selection and recovery of transgenic events, and still fall within
the scope of the present invention.
Nutrient media is prepared as a liquid, but this may be solidified
by adding the liquid to materials capable of providing a solid
support. Agar is most commonly used for this purpose. Bactoagar,
Hazelton agar, Gelrite, and Gelgro are specific types of solid
support that are suitable for growth of plant cells in tissue
culture. Some cell types will grow and divide either in liquid
suspension or on solid media.
Recipient cell targets include, but are not limited to, meristem
cells, callus, immature embryos and gametic cells such as
microspores pollen, sperm and egg cells. Any cell from which a
transgenic plant, including a fertile transgenic plant, may be
regenerated may be used in certain embodiments. For example,
immature embryos may be transformed followed by selection and
initiation of callus and subsequent regeneration of transgenic
plants. Direct transformation of immature embryos obviates the need
for long term development of recipient cell cultures. Meristematic
cells (i.e., plant cells capable of continual cell division and
characterized by an undifferentiated cytological appearance,
normally found at growing points or tissues in plants such as root
tips, stem apices, lateral buds, etc.) may also be used as a
recipient plant cell. Because of their undifferentiated growth and
capacity for organ differentiation and totipotency, a single
transformed meristematic cell could be recovered as a whole
transformed plant.
Somatic cells are of various types. Embryogenic cells are one
example of somatic cells which may be induced to regenerate a plant
through embryo formation. Non-embryogenic cells are those which
typically will not respond in such a fashion.
Certain techniques may be used that enrich recipient cells within a
cell population. For example, Type II callus development, followed
by manual selection and culture of friable, embryogenic tissue,
generally results in an enrichment of recipient cells for use in,
for example, micro-projectile transformation.
Selection in culture may be carried out following plant cell
transformation using a variety of transformation methods.
Agrobacterium transformation followed by selection is described in
the working examples below. In addition, exemplary procedures for
selection of transformed cells prepared by microprojectile
bombardment are provided as follows:
1. Tissue (suspension) is plated on filters, microprojectile
bombarded and then filters transferred to culture medium. After 2-7
days, filters are transferred to selective medium. Approximately 3
weeks after bombardment, tissue is picked from filters as separate
callus clumps onto fresh selective medium.
2. As in 1 above, except after bombardment the suspension is put
back into liquid--subjected to liquid selection for 7-14 days and
then pipetted at a low density onto fresh selection plates.
3. Callus is bombarded while sitting directly on medium or on
filters. Cells are transferred to selective medium 1-14 days after
particle bombardment. Tissue is transferred on filters 1-3 times at
2 weeks intervals to fresh selective medium. Callus is then briefly
put into liquid to disperse the tissue onto selective plates at a
low density.
4. Callus tissue is transferred onto selective plates one to seven
days after DNA introduction. Tissue is subcultured as small units
of callus on selective plates until transformants are
identified.
In certain embodiments, recipient cells are selected following
growth in culture. Cultured cells may be grown either on solid
supports or in the form of liquid suspensions. In either instance,
nutrients may be provided to the cells in the form of media, and
environmental conditions controlled. There are many types of tissue
culture media comprised of amino acids, salts, sugars, growth
regulators and vitamins. Most of the media employed in the practice
of the invention will have some similar components, while the media
can differ in composition and proportions of ingredients according
to known tissue culture practices. For example, various cell types
usually grow in more than one type of media, but will exhibit
different growth rates and different morphologies, depending on the
growth media. In some media, cells survive but do not divide. Media
composition is also frequently optimized based on the species or
cell type selected.
Various types of media suitable for culture of plant cells have
been previously described. Examples of such media are defined
below. In some embodiments, it may be preferable to use a media
with a somewhat lower ammonia/nitrate ratio such as N6 to promote
generation of recipient cells by maintaining cells in a
proembryonic state capable of sustained divisions. In certain
embodiments of the present invention, Woody Plant Medium (WPM) was
used (Lloyd and McCown, 1981).
The method of maintenance of cell cultures may contribute to their
utility as sources of recipient cells for transformation. Manual
selection of cells for transfer to fresh culture medium, frequency
of transfer to fresh culture medium, composition of culture medium,
and environment factors including, but not limited to, light
quality and quantity and temperature are all factors in maintaining
callus and/or suspension cultures that are useful as sources of
recipient cells. Alternating callus between different culture
conditions may be beneficial in enriching for recipient cells
within a culture. For example, cells may be cultured in suspension
culture, but transferred to solid medium at regular intervals.
After a period of growth on solid medium, cells can be manually
selected for return to liquid culture medium. Repeating this
sequence of transfers to fresh culture medium may be used to enrich
for recipient cells. Passing cell cultures through a 1.9 mm sieve
may also be useful to maintain the friability of a callus or
suspension culture and enriching for transformable cells when such
cell types are used.
C. Transgenic Plants
Once a transgenic cell has been selected, the cell can be
regenerated into a transgenic plant using techniques well known in
the art. The transformed plants can be subsequently analyzed to
determine the presence or absence of a particular nucleic acid of
interest contained on a DNA construct. Molecular analyses can
include, but are not limited to, Southern blots (Southern, 1975),
northern blot analysis, western blot analysis, or PCR analyses,
immunodiagnostic approaches, and field evaluations. These and other
well known methods can be performed to confirm the stability of the
transformed plants produced by the methods disclosed. These methods
are well known to those of skill in the art (Sambrook et al.,
1989).
Transgenic plants tolerant to auxin-like herbicides can be
produced. In particular, economically important plants, including
crops, fruit trees, and ornamental plants and trees that are
currently known to be injured by auxin-like herbicides can be
transformed with DNA constructs of the present invention so that
they become tolerant to the herbicide. Plants that are currently
considered tolerant to auxin-like herbicides can be transformed to
increase their tolerance to the herbicide. Examples of plants that
may in particular find use with the current invention include, but
are not limited to, alfalfa, beans, broccoli, cabbage, carrot,
cauliflower, celery, cotton, cucumber, eggplant, lettuce, melon,
pea, pepper, pumpkin, radish, rapeseed, spinach, soybean, squash,
tomato, watermelon, corn, onion, rice, sorghum, wheat, rye, millet,
sugarcane, oat, triticale, switchgrass, and turfgrass.
Once a transgenic plant containing a transgene has been prepared,
that transgene can be introduced into any plant sexually compatible
with the first plant by crossing, without the need for ever
directly transforming the second plant. Therefore, as used herein
the term "progeny" denotes the offspring of any generation of a
parent plant prepared in accordance with the instant invention,
wherein the progeny comprises a selected DNA construct prepared in
accordance with the invention. A "transgenic plant" may thus be of
any generation. "Crossing" a plant to provide a plant line having
one or more added transgenes or alleles relative to a starting
plant line, as disclosed herein, is defined as the techniques that
result in a particular sequence being introduced into a plant line
by crossing a starting line with a donor plant line that comprises
a transgene or allele of the invention. To achieve this one could,
for example, perform the following steps: (a) plant seeds of the
first (starting line) and second (donor plant line that comprises a
desired transgene or allele) parent plants; (b) grow the seeds of
the first and second parent plants into plants that bear flowers;
(c) pollinate a flower from the first parent plant with pollen from
the second parent plant; and (d) harvest seeds produced on the
parent plant bearing the fertilized flower.
D. Definitions
As used herein, the term "transformed" refers to a cell, tissue,
organ, or organism into which has been introduced a foreign
polynucleotide molecule, such as a construct. The introduced
polynucleotide molecule may be integrated into the genomic DNA of
the recipient cell, tissue, organ, or organism such that the
introduced polynucleotide molecule is inherited by subsequent
progeny. A "transgenic" or "transformed" cell or organism also
includes progeny of the cell or organism and progeny produced from
a breeding program employing such a transgenic plant as a parent in
a cross and exhibiting an altered phenotype resulting from the
presence of a foreign polynucleotide molecule.
"Contacting" the transformed plant cell with a tissue culture
medium containing an auxin-like herbicide can be achieved by
culturing the plant cell in a plant tissue culture medium
containing an auxin-like herbicide.
"Tissue culture medium" refers to liquid, semi-solid, or solid
medium used to support plant growth and development in a non-soil
environment. Suitable plant tissue culture media are known to one
of skill in the art may include MS-based media (Murashige and
Skoog, 1962) or N6-based media (Chu et al., 1975) supplemented with
additional plant growth regulators such as auxins, cytokinins,
kinetin, ABA, and gibberellins. Other media additives can include
but are not limited to amino acids, macroelements, iron,
microelements, inositol, vitamins and organics, carbohydrates,
undefined media components such as casein hydrolysates, with or
without an appropriate gelling agent such as a form of agar, such
as a low melting point agarose or Gelrite.RTM. if desired for
preparing semi-solid or solid medium. Those of skill in the art are
familiar with the variety of tissue culture media, which when
supplemented appropriately, support plant tissue growth and
development and are suitable for plant transformation and
regeneration. These tissue culture media can either be purchased as
a commercial preparation or custom prepared and modified. Examples
of such media would include but are not limited to Murashige and
Skoog (1962), N6 (Chu et al., 1975), Linsmaier and Skoog (1965),
Uchimiya and Murashige (1962), Gamborg's media (Gamborg et al.,
1968), D medium (Duncan et al., 1985), McCown's Woody plant media
(McCown and Lloyd, 1981), Nitsch and Nitsch (1969), and Schenk and
Hildebrandt (1972) or derivations of these media supplemented
accordingly. Those of skill in the art are aware that media and
media supplements such as nutrients and growth regulators for use
in transformation and regeneration and other culture conditions
such as light intensity during incubation, pH, and incubation
temperatures can be optimized for a plant of interest.
"Auxin-like herbicides" are also called auxinic or growth regulator
herbicides or Group 4 herbicides (based on their mode of action).
These herbicides mimic or act like the natural plant growth
regulators called auxins. Auxins include natural hormones such as
indole acetic acid and naphthalene acetic acid, both of which are
responsible for cell elongation in plants. The mode of action of
the auxinic herbicides is that they appear to affect cell wall
plasticity and nucleic acid metabolism, which can lead to
uncontrolled cell division and growth. The group of auxin-like
herbicides includes four chemical families: phenoxy, carboxylic
acid (or pyridine), benzoic acid, and the newest family quinaline
carboxylic acid. Phenoxy carboxylic acids: the phenoxy herbicides
are most common and have been used as herbicides since the 1940s
when (2,4-dichlorophenoxy)acetic acid (2,4-D) was discovered. Other
examples include 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB),
2-(2,4-dichlorophenoxy) propanoic acid (2,4-DP),
(2,4,5-trichlorophenoxy)acetic acid (2,4,5-T),
2-(2,4,5-Trichlorophenoxy) Propionic Acid (2,4,5-TP),
2-(2,4-dichloro-3-methylphenoxy)-N-phenylpropanamide (clomeprop),
(4-chloro-2-methylphenoxy)acetic acid (MCPA),
4-(4-chloro-o-tolyloxy) butyric acid (MCPB), and
2-(4-chloro-2-methylphenoxy) propanoic acid (MCPP).
Pyridine carboxylic acids: the next largest chemical family is the
carboxylic acid herbicides, also called pyridine herbicides.
Examples include 3,6-dichloro-2-pyridinecarboxylic acid
(Clopyralid), 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid
(picloram), (2,4,5-trichlorophenoxy)acetic acid (triclopyr), and
4-amino-3,5-dichloro-6-fluoro-2-pyridyloxyacetic acid
(fluoroxypyr). Benzoic acids: Examples include
3,6-dichloro-o-anisic acid (dicamba) and
3-amino-2,5-dichlorobenzoic acid (choramben). Quinaline carboxylic
acids: the fourth and newest chemical family of the auxinic
herbicides is the quinaline carboxylic acid family. Example
includes 3,7-dichloro-8-quinolinecarboxylic acid (quinclorac). This
herbicide is unique in that it also will control some grass weeds,
unlike the other auxin-like herbicides which essentially control
only broadleaf or dicotyledonous plants. The other herbicide in
this category is 7-chloro-3-methyl-8-quinolinecarboxylic acid
(quinmerac).
"Auxin-like herbicide effect" means injury symptoms caused by
auxin-like herbicides. These include epinastic bending and twisting
of stems and petioles, leaf cupping and curling, and abnormal leaf
shape and venation. All of these herbicides translocate, with some
translocating more than others. Some of these herbicides have soil
activity and some can persist in soil for fairly long time periods.
Due to their effect, they are used widely on many crops including
small grain cereals, corn, rice, and other grass crops, turf,
range-land, non-crop, and industrial sites.
"Selecting" the transformed plant cell that is tolerant to an
auxin-like herbicide can be achieved by methods described in the
present invention. Briefly, at least some of the plant cells in a
population of starting cells are transformed with a DNA construct
containing a DMO-encoding polynucleotide molecule. The resulting
population of plant cells is placed in a culture medium containing
an auxin-like herbicide at a concentration selected so that
transformed plant cells will grow, whereas untransformed plant
cells will not. Suitable concentrations of an auxin-like herbicide
can be determined empirically. Before selecting, explants may be
cultured on a medium without auxin-like herbicide. Such medium is
called delay medium. Explants may be placed on delay medium to
allow for some time to grow before being placed on the selection
medium. Selection regimes could be optimized depending upon a
particular auxin-like herbicide and the explant system. Often
multiple steps of selection are used and varying amounts of
selection agent can be used in each step.
"Tolerant" means that transformed plant cells are able to survive
and regenerate into plants when placed in a culture medium
containing a level of an auxin-like herbicide that prevents
untransformed cells from doing so. "Tolerant" also means that
transformed plants are able to grow after application of an amount
of an auxin-like herbicide that inhibits the growth of
untransformed plants.
EXAMPLES
The following examples are included to illustrate embodiments of
the invention. It should be appreciated by those of skill in the
art that the techniques disclosed in the examples that follow
represent techniques discovered by the inventor to function well in
the practice of the invention. However, those of skill in the art
should, in light of the present disclosure, appreciate that many
changes can be made in the specific embodiments which are disclosed
and still obtain a like or similar result without departing from
the concept, spirit and scope of the invention. More specifically,
it will be apparent that certain agents which are both chemically
and physiologically related may be substituted for the agents
described herein while the same or similar results would be
achieved. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the invention as defined by the appended
claims.
Example 1
Preparation of DMO-Encoding Polynucleotide Constructs
Several binary vectors were prepared for testing the ability of
DMO-encoding polynucleotide molecules to allow selection of
transformed soybean cells. Genetic elements used for preparing the
binary vectors are given in Table 1 and include a CaMV 35S promoter
and enhancer (U.S. Pat. Nos. 5,322,938; 5,352,605; 5,359,142; and
5,530,196); GmHsp untranslated leader from the Hsp17.9 gene of
Glycine max (U.S. Pat. No. 5,659,122); AvGFPI coding region for the
first 126.3 amino acids of the GFP protein from Aequorea victoria
(U.S. Pat. Nos. 5,491,084; 6,146,826) with a serine to threonine
change at amino acid 65 and optimized for plant expression; a StLS1
second intron from the LS1 gene of Solanum tuberosum (Eckes et al.,
1986); an AvGFPII coding region for the last 112.6 amino acids of
the GFP protein from Aequorea victoria (U.S. Pat. Nos. 5,491,084;
6,146,826) optimized for plant expression; a T-Atnos 3'
untranslated region of the nopaline synthetase gene from
Agrobacterium tumefaciens (Rojiyaa et al., 1987, GenBank Accession
E01312); a FMV Figwort Mosaic Virus 35S promoter (U.S. Pat. Nos.
6,051,753; 5,378,619); a PhDnaK untranslated leader from Hsp70 gene
of Petunia hybrida (U.S. Pat. No. 5,362,865); and an AtRbcS4 (CTP1)
coding region for Arabidopsis SSU1A transit peptide. The latter
element includes the transit peptide, 24 amino acids of the mature
protein, and a repeat of the last 6 amino acids of the transit
peptide (U.S. Pat. No. 5,728,925). Also used were an AtShkG (CTP2)
coding region for Arabidopsis thaliana
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) transit
peptide. This element varies from the wild-type sequence (Klee et
al., 1987) in that the last codon was changed from GAG (glutamic
acid) to TGC (cysteine). An AtShkGzmcodon (CTP2syn) element was
used which is the same as AtShkG (CTP2) but optimized for plant
expression using Zea mays codons (see SEQ ID NO:14 of WO04009761).
A PmDMOCys112Atcodon region for dicamba monooxygenase from
Pseudomonas maltophilia was used (US Patent Application
20030115626) having a cysteine at 112 position and optimized for
dicot expression using Arabidopsis thaliana codons (SEQ ID NOs: 1,
3, 7). Also used for construct design were a PmDMOTrp112Atcodon
coding region for dicamba monooxygenase from Pseudomonas
maltophilia (US patent application 20030115626) having a tryptophan
(Trp) at 112 position and optimized for dicot expression using
Arabidopsis thaliana codons (SEQ ID NOs: 5, 9); a PsRbcS2: 3'
polyadenylation region from the RbcS2-E9 gene of Pisum sativum
(Coruzzi et al., 1984); an AtAnt1 promoter/intron and leader of
adenine nucleotide translocator 1 gene from Arabidopsis thaliana;
an AtaroA-CP4 coding region for non-naturally occurring aroA-CP4
(U.S. Pat. No. 5,633,435) engineered for expression in plants; a
TEV 5' untranslated leader from the Tobacco Etch RNA virus
(Carrington and Freed, 1990); a PsRbcS chloroplast transit peptide
from ribulose 1,5-bisphosphate carboxylase small subunit of pea and
first 24 amino acids of the mature rubisco protein (Coruzzi et al.,
1984); a P-Atnos promoter for nopaline synthetase of Agrobacterium
tumefaciens pTiT37 (GenBank Accession V00087; Depicker et al, 1982;
Bevan et al., 1983); a L-At.nos 5' untranslated region from the
nopaline synthetase gene of Agrobacterium tumefaciens pTiT37
(GenBank Accession V00087; Bevan et al., 1983), and a PClSV
promoter for the full length transcript of peanut chlorotic streak
virus. The latter element has a duplication of 179 nt in direct
repeats with 6 nt between the repeat followed by the 70 by region
containing the TATA box (U.S. Pat. No. 5,850,019). Different CTPs
and DMO-encoding polynucleotide molecule variants are summarized in
Table 2.
TABLE-US-00001 TABLE 1 Genetic elements used for constructing
T-DNAs Expression Unit 1 Expression Unit 2 Pro- Pro- Construct
moter 5'UTR CR Intron CR PolyA moter 5' UL TS CR PolyA pMON73690
CaMV GmHsp AvGFPI StLS1 AvGFPII T-Atnos FMV PhDnaK None Pm-DMOCy-
s.sub.112Atcodon PsRbcS2 pMON73691 CaMV GmHsp AvGFPI StLS1 AvGFPII
T-Atnos FMV PhDnaK AtShkG Pm-DMO- Cys.sub.112Atcodon PsRbcS2
pMON73696 CaMV GmHsp AvGFPI StLS1 AvGFPII T-Atnos FMV PhDnaK
AtRbcS4 Pm-DM- OTrp.sub.112Atcodon PsRbcS2 pMON73698 CaMV GmHsp
AvGFPI StLS1 AvGFPII T-Atnos FMV PhDnaK AtRbcS4 Pm-DM-
OCys.sub.112Atcodon PsRbcS2 Pro- Pro- 5' moter 5'UR TS CR moter UL
TS CR PolyA pMON73724 AtAnt1 AtAnt1 At.ShkG At.aroA-CP4 PsRbcS2
PC1SV TEV AtShkGZ- PmD- MOTrp.sub.112 Atnos mcodon Atcodon First
T-DNA Second T-DNA Promoter 5'UTR TS CR PolyA Promoter 5' UL TS CR
PolyA pMON58498 PC1SV TEV PsRbcS PmDMO PsRbcS2 FMV PhDnaK AtShkG
AtaroA-CP4 PsRb- cS2 Cys.sub.112 pMON84254 PC1SV TEV PsRbcS PmDMO
PsRbcS2 P-Atnos L-Atnos None Sh.bar T-Atn- os Cys.sub.112 Key:
5'UTR: 5' untranslated region; CR: coding region; Poly A:
polyadenylation region; and TS: transit sequence.
TABLE-US-00002 TABLE 2 Chloroplast transit peptides and
DMO-encoding polynucleotides used in binary vectors. CTP SEQ
Predicted aa Predicted aa at Construct variant DMO variant ID
Length at position 2 position 112 pMON73690 None DMO-Cys.sub.112 1
1023 Ala Cys and codon optimized for dicots pMON73691 CTP2
DMO-Cys.sub.112 3 1023 Leu Cys and codon optimized for dicots
pMON73696 CTP1 DMO-Trp.sub.112 5 1023 Leu Trp and codon optimized
for dicots pMON73698 CTP1 DMO-Cys.sub.112 3 1023 Leu Cys and codon
optimized for dicots pMON58498 PsRbcS DMO-Cys.sub.112 7 1023 Ala
Cys pMON84254 PsRbcS DMO-Cys.sub.112 7 1023 Ala Cys pMON73724
CTP2Zm DMO-Trp.sub.112 9 1023 Ala Trp and codon optimized for
dicots
In the case of pMON73690, pMON73691, pMON73696, and pMON73698, the
DMO-encoding polynucleotide molecule was linked to the screenable
marker GFP and provided on the same T-DNA to show that the
DMO-encoding polynucleotide molecule can be used with another gene.
In case of pMON58498, pMON84254, and pMON73724, the DMO-encoding
polynucleotide molecule was unlinked from the other transgene
(selectable marker or agronomic trait gene) by separating them on
two T-DNAs.
Example 2
Development of Selection Method
Mature seeds of soybean [Glycine max (L.) Merrill] cv. A3525 were
imbibed, sterilized, and germinated at room temperature as set
forth below. Other examples of soybean genotypes that can readily
be used include, but are not limited to, Jack, Williams, Bert,
Thorne, Granite, Lambert, Chapman, and Kunitz. Briefly, dry seeds
(about 770 g) were soaked for 3 min in 2 L of 200 ppm sodium
hypochlorite solution made from commercially available Clorox. The
solution was drained and the seeds were set side for about 2 h.
About 2 L of bean sterilization/germination medium was then added
to the seeds. After about 9-10 h, seeds were ready for hand
excision of explants. The bean germination medium contained the
following in mg/L--NH.sub.4NO.sub.3: 240, KNO.sub.3: 505,
CaCl.sub.2.2H.sub.2O: 176, MgSO.sub.4.7H.sub.2O: 493,
KH.sub.2PO.sub.4: 27, H.sub.3BO.sub.3: 1.86,
Na.sub.2MoO.sub.4.2H.sub.2O: 0.216, MnSO.sub.4.H.sub.2O: 5.07,
ZnSO.sub.4. 7H.sub.2O: 2.58, FeSO.sub.4.7H.sub.2O: 2.502, KI:
0.249, Na.sub.2EDTA. 2H.sub.2O: 3.348, CuSO.sub.4.5H.sub.2O:
0.0008, CoCl.sub.2.6H.sub.2O: 0.0008, B1: 1.34, B3: 0.5, B6: 0.82,
Bravo (75% WP; Diamond Shamrock Company, Cleveland, Ohio): 30,
Captan (50% WP; Micro Flo Company, Lakeland, Fla.): 30, Cefotaxime:
125, and Sucrose: 25000, pH 5.8).
For machine excision of the explants, seeds were treated with 2 L
of 200 ppm sodium hypochlorite solution for 15 min. After draining
the solution the seeds were rinsed with 2 L of sterile distilled
water for 1 min. The machine and method for mechanical excision are
described in the US Patent Appln. Pub. 20050005321. Briefly,
imbibed seeds were run through three sets of rollers in the
machine, with sterile distilled water running over them, and
crushed. A mixture of cotyledons, seed coats and the explants
(embryo axis) is collected and sieved by either hand or by using an
auto-sieving device to recover the explants. The explants were
rinsed with 0.05% ethanol for 1 min, followed by two rinses with
sterile distilled water for removing more debris.
The binary vectors described above were mobilized into disarmed
Agrobacterium tumefaciens strain C58 (ABI). Agrobacterium inoculum
for infection was prepared as follows: 250 ml of LB medium
(Luria-Bertani; Difco, Detroit, Mich.) containing 50 mg/l kanamycin
(Sigma, St. Louis, Mo.) and 75 mg/l spectinomycin (Sigma, St.
Louis, Mo.) was inoculated with 0.5 ml of Agrobacterium stock in
glycerol (Acros Organics, Geel, Belgium) and was shaken at 200 rpm
at 28.degree. C. for approximately 20-22 h until the OD.sub.660
reached 0.8 to 1.0. The Agrobacterium broth was then centrifuged
for 25 min at 3500 rpm (about 3565 g) at 2-4.degree. C. After
removing the supernatant, the Agrobacterium pellet was re-suspended
in inoculation medium containing .times. of the macro nutrients,
1/10.times. of the micro nutrients and vitamins of Gamborg's B5
medium, supplemented with 3.9 g/l MES (Sigma, St. Louis, Mo.), and
30 g/l glucose (PhytoTechnology Laboratories, Shawnee Mission,
Kans.), pH 5.4. Lipoic acid (Sigma, St. Louis, Mo.) was added to
the Agrobacterium suspension to a final concentration of 0.25 mM
after the density of the Agrobacterium cell suspension was adjusted
to an OD.sub.660 of 0.30 to 0.35.
Agrobacterium-infection and co-cultivation of the explants were
conducted as follows: about 100 excised explants were dispensed
into the lid of a sterile plastic culture vessel PLANTCON (MP
Biomedicals, LLC, Irvine, Calif.). Five ml of Agrobacterium
inoculum was added to the explants in each PLANTCON lid. The
explants were then sonicated for 20 sec in a sonicator (Ultrasonic
Multi Cleaner, Model W-113, Honda, Japan). One piece of Whatmann #1
filter paper (Whatman Inc., Clifton, N.J.) cut to the size of the
PLANTCON bottom was placed in the bottom part of the PLANTCON. The
explants were transferred from the lid onto the filter paper with
the Agrobacterium inoculum. The PLANTCON s were then incubated in a
Percival incubator at 16 h light (at about 85-90 .mu.E) and 8 h
dark photoperiod and at 23.degree. C. for 2 to 4 d for
co-cultivation.
After a co-cultivation period of 2-4 days, explants were first
cultured on a medium without dicamba (delay medium) for 3-5 d
before being transferred to the selection medium with dicamba.
Until transfer from delay medium to selection medium, the explants
were kept in the same PLANTCON used for co-cultivation, but 10 or
12 ml of the delay medium was added to each PLANTCON.
Alternatively, the explants were transferred to new PLANTCONs, each
containing one piece of autoclaved felt (Jo-Ann Fabrics &
Crafts, Madison, Wis.)) and 30 ml of the delay medium. The delay
medium contained modified wood plants medium (Lloyd and McCown,
1981) supplemented with 1 or 5 mg/l BAP (6-benzyl Amino Purine),
200 mg/l carbenicillin (Phyto Technology Laboratories, Shawnee
Mission, Kans.), 200 mg/l cefotaxime (Hospira, Lake Forest, Ill.)
and 100 mg/l ticarcillin (Duchefa, The Netherlands). BAP may help
maintain the auxin-cytokinin ratio as dicamba is an auxin
herbicide, and promote production of multiple shoots from the
apical meristem. Other suitable cytokinins that can be useful in
practicing the present invention include: Adenine cytokinins (e.g.,
kinetin, zeatin, benzyl adenine (i.e. 6-Benzyl aminopurine),
adenine and Phenylurea cytokinins (e.g., N,N'-diphenylurea), and
Thidiazuron (TDZ).
Selection was conducted in a liquid or on a semi-solid medium. The
selection medium was the delay medium absent BAP and contained
different concentrations of dicamba. For selection in liquid
medium, 50 or 60 ml of the selection medium and one piece of foam
sponge (Wisconsin Foam Products, Madison, Wis.) having 5 parallel
slits were placed in each Plantcon. Twenty-five explants were
implanted into the slits in an upward position such that apical
meristem faced upward. Every two to three weeks, old medium was
replaced with the fresh medium.
Semi-solid medium was prepared by adding 4 g/l AgarGel (Sigma, St.
Louis, Mo.) to the liquid medium. For selection on semi-solid
selection medium, the explants were individually implanted into the
medium in PLANTCONs. At the late stage of the selection and shoot
development (approximately 4 weeks on the selection medium), 20 ml
of the liquid selection medium was optionally overlaid on the
semi-solid medium. Elongated shoots with expanded trifoliate
foliage leaves started to develop after the explants had been
cultured on the selection medium for about 4 to 5 weeks. These
tolerant shoots were detached from the original explants when they
were about and over 2 cm long and transferred to the liquid or
semi-solid root induction medium.
The medium for root induction was the same as for shoot development
and was also supplemented with dicamba to reduce the frequency of
escapes. Alternatively, Bean Rooting Medium (BRM) supplemented with
0.01 mg/l dicamba was used for root induction. This medium
contained 1/2 strength of MS salts, MS vitamins, 100 mg/l inositol,
100 mg/l cysteine, 30 mg/l sucrose and 100 mg/l ticarcilin and was
solidified with 8 g/l washed agar. For root induction in the liquid
medium, enough small glass beads (Inotech Biosystems International
Inc., Dottikon, Switzerland) and 60 ml of the rooting medium were
placed in each PLANTCON such that the medium and beads were at the
same level. Up to nine detached shoots were inserted into the beads
for liquid root induction or in semi-solid medium in each PLANTCON.
Almost all shoots could produce roots on the rooting medium in 1-2
weeks (FIG. 5). However, only those shoots in which the existing
and newly developed leaves remained expanded and grew vigorously
were transferred to soil for growing to maturity. All cultures were
kept under fluorescent light with a photoperiod of 16 h with light
intensity of about 20-70 .mu.E at 27-28.degree. C. until R.sub.0
plants were transferred to the soil.
In one study, soybean cells transformed with pMON73691 were
selected on 0.01 to 0.1 mg/l of dicamba in selection medium (FIGS.
1 A & B; FIG. 4). Shoots coming out of explants grown on
selection medium with 0.05 or 0.1 mg/l dicamba did not have much
growth and eventually bleached out and no tolerant shoots were
obtained. However, in selection medium containing 0.01 mg/l
dicamba, 30 dicamba-tolerant shoots were harvested from 800
explants. Twelve of these formed roots on rooting medium and were
transferred to the soil. Ten of these were tested for DMO-encoding
polynucleotides and seven were found to be positive. At a dicamba
level of 0.02 mg/l, few tolerant shoots were harvested. These
results suggested that a dicamba concentration of 0.01 mg/l or
lower was most efficacious for selecting dicamba-tolerant shoots.
This level could readily be altered by one skill in the art for
particular studies, however, depending upon the nature of the
explant, construct, and other variables.
In order to demonstrate the selection of tolerant shoots containing
a linked gene, a plant expressible DMO-encoding nucleic acid
coupled to a plant expressible GFP-encoding nucleic and introduced
into cells following selection. The cultures were examined for GFP
expression 45 days after inoculation (DAI). GFP-positive small buds
were observed on several explants, suggesting that these buds
originated from cells transformed with the linked DMO-encoding
polynucleotide molecule (FIG. 2). Several of these buds developed
into GFP positive shoots and were positive for GFP gene (FIGS. 3,
6). These results demonstrated that a DMO-encoding polynucleotide
molecule can be used as a selectable marker and be used for
recovery of transformants containing and expressing a linked gene.
Confirmation that the GFP transgene was inherited in the progeny
was found by self-pollinating R.sub.0 plants transformed with
pMON73691. Immature R.sub.1 seeds (about 4 mm in length) were
collected and cut into two halves to expose the cotyledon tissue.
GFP expression was detected in the cotyledon tissue of seeds under
a dissecting microscope equipped with fluorescent light.
Rooting was also accomplished in Oasis Growing Medium i.e. plugs
(Smithers-Oasis North America, Kent, Ohio, USA). A total of 102
Dicamba-selected shoots were inserted into Oasis plugs for inducing
roots. The plugs were surrounded by a liquid medium containing 0.01
mg/l dicamba. The shoots in the plugs were kept in culture room at
28.degree. C. and 16-h light. Thirty shoots developed roots and
appeared to be resistant to dicamba showing relatively expanded new
leaves. The plants with roots were tested by invader assay and 19
plants were found to contain both DMO and GUS genes. The escape
rate on the liquid medium was about 33%, which was much lower than
the 53% escape rate when the roots were induced in the semi-solid
medium. The negative phenotypes of the shoots i.e., cupping leaves
could be seen sooner in the liquid selection medium than in the
semi-solid medium. This suggested that rooting in the liquid
selection medium could be a more efficient method to eliminate
escapes.
Example 3
Molecular Analysis of Transformed Soybean Plants
In order to confirm that the dicamba tolerant plants obtained were
the result of transfer of DMO-encoding polynucleotides, leaf tissue
was collected from each R.sub.0 or R.sub.1 plant, DNA was
extracted, and the presence of the DMO-encoding polynucleotide was
confirmed by Invader.TM. technology (Third Wave Technologies,
Madison, Wis.) and Southern blot analysis using non-radioactive
probe kit from Roche (Indianapolis, Ind.).
For the Invader assay, the primers used were: primary probe
5'-acggacgcggag ATGCTCAACTTCATCGC-3' (SEQ ID NO: 13) and Invader
oligo 5'-TCCGCTGGAACAAGGTGAGCGCGT-3' (SEQ ID NO: 14). The sequence
in lower case letters in the primary probe is the 5' flap sequence
which is cleaved and is not complimentary to the target
sequence.
For Southern blot analysis a DNA fragment of 897 bp was used to
prepare the probe. The forward primer 5'-GTCGCTGCCCTGCTTGATATT-3'
(SEQ ID NO: 15) and the reverse primer 5'-CGCCGCTTCTAGTTGTTC-3'
(SEQ ID NO: 16) were used to amplify the 897 bp DNA fragment. A
total of 12 rooted shoots selected on 0.01 mg/l dicamba shoots were
transferred to soil. Ten plants were assayed by Invader and/or
Southern analysis. Seven of these showed the presence of the
DMO-encoding nucleic acid (Table 3). Several of these were also
positive for GFP-encoding polynucleotides confirming the ability to
use the DMO-encoding nucleic acid as a selectable marker for
recovery of transformants containing a linked gene.
TABLE-US-00003 TABLE 3 Testing of R0 plants for DMO-encoding
nucleic acid by Invader and/or Southern Analysis. Plant Name Origin
(Exp-Trt) Invader Southern GM_A4755D 533-1 + + GM_A4756D 533-1 - -
GM_A4757D 533-1 - - GM_A4758D 533-1 - - GM_A4763D 533-1 + +
GM_A4759D 534-1 + + GM_A4760D 534-1 - + GM_A4761D 534-1 + +
GM_A4764D 534-1 N/T + GM_A5087D 534-1 N/T +
Example 4
Selection of Dicamba-Tolerant Plants Transformed with DMO-Encoding
Polynucleotide Molecule Variants
Two DMO-encoding polynucleotide molecule variants were used to
obtain dicamba tolerant plants. The first variant had cysteine at
amino acid position 112 (DMOCys.sub.112; pMON73698) and the second
had tryptophan at amino acid position 112 (DMOTrp.sub.112;
pMON73696). Selection using both variants resulted into dicamba
tolerant shoots (FIG. 7). Following selection and shoot and root
induction, the rooted plants were moved to soil for growing to
maturity and assayed by Invader.TM. and/or Southern analysis for
the presence of DMO-encoding nucleic acid. Several plants
transformed with pMON73696 were found to be positive for the DMO
gene in R.sub.0 and R.sub.1 generation indicating germline
transformation using the method of the present invention.
TABLE-US-00004 TABLE 4 Selection of dicamba-tolerant shoots
transformed with DMO-encoding nucleic acid variants. # Tolerant #
Rooted # plants # shoots shoots moved with DMO gene Medium
Construct Explants harvested to soil (# plants assayed) Liquid
73696 (DMOTrp.sub.112) 1022 6 1 0 73698 (DMOCys.sub.112) 869 0 0 0
73696 (DMOTrp.sub.112) 1200 25 9 3 73698 (DMOCys.sub.112) 1200 0 0
0 Semisolid 73696 (DMOTrp.sub.112) 1536 94 50 28 (47) 73698
(DMOCys.sub.112) 1845 3 0 0 (0) 73696 (DMOTrp.sub.112) 450 27 18 10
(16) 73698 (DMOCys.sub.112) 475 0 0 0 (0)
Example 5
Selection of Dicamba-Tolerant Plants Transformed with DMO-Encoding
Polynucleotide Molecules Combined with Different Chloroplast
Transit Peptides
It is known that different chloroplast transit peptides (CTPs)
target a foreign polypeptide to chloroplasts with different
efficiencies. The effect of different types of CTPs was therefore
tested by transforming soybean explants with DMO-encoding
polynucleotide molecules targeted either by CTP2 (pMON73691) or
CTP1 (pMON73698) or not targeted to chloroplasts (pMON73690). As
shown in Table 5, in general shoots were harvested with constructs
containing either CTP2 or CTP1 (also see FIG. 7). The rooted plants
were moved to soil for growing to maturity and assayed by
Invader.TM. and/or Southern analysis for the presence of
DMO-encoding nucleic acid. Several plants transformed with
pMON73691 were found to be positive for the DMO gene in R.sub.0 and
R.sub.1 generation indicating germline transformation using the
method of the present invention.
Several transgenic plants carrying either a PClSV/RbcS/DMO-Wdc/Nos
or PClSV/CTP2nat/DMO-Cnative/Nos expression unit were also found to
be tolerant to a dicamba treatment at the rate of 1 lb/A (Clarity,
BASF) at V3-4 when analysed 18 DAT in a greenhouse study.
TABLE-US-00005 TABLE 5 Selection of dicamba tolerant plants
transformed with DMO-encoding nucleic acid combined with different
chloroplast transit peptides. # tolerant # rooted # plant with Exp-
# shoots shoots moved DMO gene (# Trt Construct Explants harvested
to soil plants assayed) 576-1 73691 (CTP2/DMOCys.sub.112) 1350 74
14 10 (14) 625-3 73691 (CTP2/DMOCys.sub.112) 500 17 6 4 (5) 576-2
73698 (CTP1/DMOCys.sub.112) 1050 22 1 1 (1) 625-1 73690
(DMOCys.sub.112) 531 1 0 0 625-2 73690 (DMOCys.sub.112) 531 2 0
0
Example 6
Use of DMO-Encoding Polynucleotide Molecule as a Selectable Marker
in Combination with an Agronomic Trait Gene
One beneficial use of a DMO-encoding polynucleotide molecule as a
selectable marker is the recovery of transformants containing a
genetically linked gene, for example, conferring an improved
agronomic trait. This ability was demonstrated by transforming
soybean explants with pMON58498 having 2-DNAs: a first T-DNA having
a DMO-encoding polynucleotide molecule and a second T-DNA having a
CP4 gene for glyphosate tolerance. Transgenic plants selected on
semi-solid medium were transferred to soil and assayed by
Invader.TM. and/or Southern analysis to show the presence of DMO
and CP4 nucleic acids.
While both the DMO and CP4-encoding polynucleotide molecules could
be used as a selectable marker, it was shown that transformants
comprising a CP4 transgene could be selected using dicamba
selection alone. As shown in Table 6, all but one regenerated plant
from each of the two treatments had both DMO and CP4 genes. This
study therefore demonstrates the ability to use DMO as a selectable
marker for the recovery of agronomic genes. It is understood that
any gene that is genetically linked to a selectable DMO marker as
introduced into a genome, e.g., present within 50 cM, can be
selected in this manner and that such genes need not necessarily be
introduced on the same vector.
TABLE-US-00006 TABLE 6 Use of DMO as a selectable marker in
combination with an agronomic trait gene. # plants # plants BAP in
with DMO with CP4 delay # tolerant # shoots gene gene medium #
shoots rooted (# plants (# plants Exp-Trt (mg/l) Explants harvested
in soil assayed) assayed) 566-1 & 1 1654 51 37 19 (37) 18 (37)
567-1 566-2 & 5 1800 35 11 3 (11) 2 (11) 567-2
Example 7
Tolerance of Plants Containing DMO-Encoding Polynucleotide Molecule
to Other Auxin-Like Herbicides
An analysis was carried out to determine whether soybean plants
having DMO-encoding polynucleotide could deactivate other
auxin-like herbicides in addition to dicamba. This was carried out
by applying various concentrations of commercially available
formulations of other auxin-like herbicides such as 2,4-D (Helena,
Collierville, Tenn.), MCPA (Agriliance, St. Paul, Minn.), triclopyr
(GARLON 3A; Dow Elanco, Indianapolis, Ind.), clopyralid (STINGER;
Dow Elanco, Indianapolis, Ind.), picloram (TORDON 22K; Dow Elanco,
Indianapolis, Ind.), or Banvel or Clarity (BASF, Raleigh, N.C.) to
DMO containing plant tissues or plants.
Transgenic soybean plants were obtained by Agrobacterium-mediated
transformation of soybean explants with a DMO-encoding
polynucleotide as described above for the events designated Events
1-4. A non-transgenic line was used as a control. Non-transgenic
and transgenic soybean seeds were planted into 3,5-inch square
plastic pots containing Redi-earth.TM. (Scotts-Sierra Horticultural
Products Co., Marysville, Ohio). The pots were placed on capillary
matting in 35 inch.times.60 inch fiberglass watering trays for
overhead and/or sub-irrigation for the duration of the test period
so as to maintain optimum soil moisture for plant growth. The pots
were fertilized with Osmocote (14-14-14 slow release; Scotts-Sierra
Horticultural Products Co., Marysville, Ohio) at the rate of 100
gm/cu.ft. to sustain plant growth for the duration of greenhouse
trials. The plants were grown in greenhouses at
27.degree./21.degree. C. day/night temperature, with relative
humidity between 25%-75% to simulate warm season growing conditions
of late spring. A 14 h minimum photoperiod was provided with
supplemental light at about 600 .mu.E as needed.
All herbicide applications were made with the track sprayer using a
Teejet 9501E flat fan nozzle (Spraying Systems Co, Wheaton, Ill.)
with air pressure set at a minimum of 24 psi (165 kpa). The spray
nozzle was kept at a height of about 16 inches above the top of
plant material for spraying. The spray volume was 10 gallons per
acre or 93 liters per hectare. Applications were made when plants
had reached V-3 stage. All trials were established in a randomized
block design (randomized by rate) with 4 to 6 replications of each
treatment depending on plant quality, availability and to account
for any environmental variability that may have occurred within the
confines of each greenhouse.
All treated plants in greenhouse trials were visually assessed at
about 4, 14, 18, and 21 days after treatment (DAT) for injury on a
scale of 0 to 100 percent relative to untreated control plants,
with zero representing "no" injury and 100% representing "complete"
injury or death. Data were collected using a palm top computer and
analyzed using standard statistical methods. The results shown in
Table 9 clearly indicate tolerance of transgenic soybean to other
auxin-like herbicides such as 2,4-D and MCPA relative to the
non-transgenic line.
TABLE-US-00007 TABLE 7 Percentage injury relative to un-treated
controls at 25 DAT post-V3 applications of different auxin-like
herbicides to non-transgenic or transgenic soybean plants.*
Herbicide Plant/trial 280 561 1120 % injury at shown rates (g
ae/ha**) at 21 DAT Dicamba (Clarity) Non-transgenic 100 100 Event 1
0.0 1.2 Event 2 0.0 1.7 Event 3 0.0 0.7 Event 4 0.0 1.5 Dicamba
(Banvel) Non-transgenic 100.0 100.0 Event 1 0.0 1.5 Event 2 0.0 0.7
Event 3 0.0 0.5 Event 4 0.0 1.3 2,4-D Non-transgenic 86.8 100.0
100.0 Event 1 58.3 75.0 100.0 Event 2 64.2 94.7 100.0 Event 3 40.0
85.0 100.0 Event 4 45.8 84.2 100.0 MCPA Non-transgenic 93.0 98.3
100.0 Event 1 72.5 99.3 100.0 Event 2 55.0 95.0 99.7 Event 3 55.0
95.8 100.0 Event 4 88.3 98.8 100.0 LSD 16.3 10.6 3.7 % injury shown
rates (g ae/ha**) at 14 DAT Triclopyr Non-transgenic 86.7 97.3 98.7
Event 1 88.3 95.7 99.3 Event 2 86.7 98.7 99.3 Event 3 86.7 94.0
96.3 Event 4 90.8 98.0 99.2 Clopyralid Non-transgenic 99.3 100.0
100.0 Event 1 99.2 100.0 100.0 Event 2 98.2 99.7 100.0 Event 3 99.3
100.0 100.0 Event 4 99.7 100.0 100.0 Picloram Non-transgenic 99.3
100.0 100.0 Event 1 99.7 100.0 100.0 Event 2 99.3 100.0 100.0 Event
3 99.3 99.7 100.0 Event 4 99.3 100.0 100.0 LSD 2.9 1.8 1.4 *The %
injury was represented as ANOVA mean comparisons. **grams of active
acid equivalent/hectare
This example shows that transgenic soybean plants exhibit tolerance
to other auxin-like herbicides, indicating a likely common
deactivation mechanism for dicamba and other auxin-like herbicides
such as 2,4-D and MCPA. In case of triclopyr, chlopyralid, and
picloram, the application rate of 280 g ae/ha appeared too
stringent in this study and thus lower concentrations may be
desired in a most settings to reduce plant damage. The results
indicate that auxin-like herbicides may be used for selecting plant
cells transformed with DMO-encoding polynucleotide molecules,
especially in case of plants that are very sensitive to dicamba,
for example, cotton. The appropriate concentration of the
auxin-like herbicide for selection under a given set of conditions
may be optimized using a test grid of treatments followed by
observation of treated plant tissues. An example of such a grid
analyzes the effect of concentrations of from about 0.001 mg/l to
about 10 mg/l, including 0.01, 0.1, 1.0, 2.0, and 5.0 mg/L.
Another auxin-like herbicide Butyrac 200 (2,4-DB; Albaugh) was also
tested on transgenic soybean plants carrying a DMO gene for testing
the plants tolerance to it. The herbicide was applied as a
post-emergence treatment at three application rates on two
transgenic soybean events and compared with a non-transgenic line
for total crop injury across all three application rates: 280 g/ha
(0.25 lb/a), 561 g/ha (0.5 lb/a) and 841 g/ha (0.75 lb/a) (see
Table 8). Both transgenic soybean lines showed low level of
tolerance to 2,4-DB. This example demonstrates that dicamba
tolerant soybean is also tolerant to low levels of 2,4-DB and
should be useful in managing damage from spray drift from the same
or neighboring fields to prevent crop loses, and would exhibit
tolerance to residual levels of 2,4-DB following incomplete washing
of herbicide delivery equipment.
TABLE-US-00008 TABLE 8 Percentage injury relative to the untreated
control at 16 DAT by the application of 2,4-DB to non-transgenic or
transgenic soybean plants. % injury at shown rates (g ae/ha) at 16
DAT Herbicide Plant 280 561 1120 2,4-DB Non-transgenic 59.2 70.0
79.2 (Butyrac 200) NE3001 462-1-21 25.0 43.3 75.8 469-13-19 18.3
37.5 70.0
Example 8
Use of DMO Gene as a Selectable Marker Against Other Auxin-Like
Herbicides
Freshly isolated soybean explants (mature embryo axes without
cotyledons) were inoculated with Agrobacterium strain ABI harboring
pMON73691 (containing DMO and GFP genes). After 3-d co-culture with
Agrobacterium at 23.degree. C. and a photoperiod of 16-h light and
8-h dark, the explants were cultured in liquid delay medium which
contained modified woody plant medium supplemented with 5 mg/l BAP,
200 mg/l carbenicillin, 200 mg/l cefotaxime and 100 mg/l
ticarcillin. The explants were in the delay medium for 4 days. They
were then transferred to liquid selection medium in PLANTCONs. The
selection medium was the same as the delay medium except of
addition of various levels of 2,4-D (0.01, 0.1, 1.0 or 2.0 mg/L) or
0.01 mg/L dicamba as shown in Table 9 below. Each PLANTCON
contained 60 ml of the selection medium and one piece of foam
sponge with 5 slits. Twenty-five explants were evenly inserted into
the slits. The cultures were maintained at 28.degree. C. and a
photoperiod of 16-h light and 8-h dark, and were examined
periodically under a sterile microscope equipped for detecting GFP
expressing tissues. At 48 days after inoculation (DAI),
GFP-expressing (GFP+) buds or young shoots were observed on number
of explants in the treatments with 0.01 mg/L dicamba, 0.01, 0.1 or
1.0 mg/L 2,4-D, but not on the explants treated with 2 mg/L 2,4-D.
Extensive callus development was observed on the explants in
treatments with 1 or 2 mg/L 2,4-D. In the treatment with 0.01 or
0.1 mg/L 2,4-D, the explants had extensive shoot growth, and a few
had elongated GFP+ shoots.
TABLE-US-00009 TABLE 9 Summary of experiment using DMO as a
selectable marker and 2,4-D as the selective agent. # Explants
Selective agent and # Explants w/GFP + buds/young Treatment #
concentration inoculated shoots at 48DAI 710-1 0.01 mg/L dicamba
375 28 (control) 710-2 0.01 mg/L 2,4-D 375 33 710-3 0.1 mg/L 2,4-D
375 19 710-4 1 mg/L 2,4-D 375 4 710-5 2 mg/L 2,4-D 375 0
Example 9
Selection of Dicamba-Tolerant Plants Transformed with DMO-Encoding
Polynucleotide without a Delay Step
Transgenic plants with a DMO gene without a delay-to-selection step
were produced in three studies. As an example, explants were
infected and co-cultivated with Agrobacterium harboring pMON73696.
After the co-culture period, the explants were cultured on liquid
medium containing 5 mg/l BAP and 0.01 mg/l dicamba for 4 day, and
then transferred onto to the liquid or semi-solid selection medium
with 0.01 mg/l dicamba. As shown in Table 10, dicamba tolerant
shoots could be obtained from the treatments (717-2 and 757-2) that
utilized selection immediately after co-culture with
Agrobacterium.
TABLE-US-00010 TABLE 10 Selection of dicamba-tolerant plants
transformed with DMO-encoding polynucleotide without a delay step.
Experimental Number of days Construct # Plants # Plants assayed #
Plants Treatment delayed to selection (pMON) # Explants moved to
soil w/Invader w/the gene 717-1 4 73696 608 15 15 4 717-2 0 73696
665 14 12 6 757-1 4 73696 542 13 11 6 757-2 0 73696 542 7 7 7
Example 10
Use of DMO as a Selectable Marker for Arabidopsis
The susceptibility of Arabidopsis to different levels of dicamba
was first tested. Wild type Arabidopsis var. Columbia seeds were
plated on plant tissue culture medium containing various levels of
dicamba. FIG. 8 shows that wild type Arabidopsis was quite
susceptible to 1.0 mg/L in the culture medium. Arabidopsis plants
were then transformed with the constructs containing DMO
polynucleotides using the floral dip method (Clough and Bent,
1998). R.sub.1 seeds were plated on the culture medium containing
up to 4 mg/L of dicamba. FIG. 9, for example, shows recovery of
dicamba tolerant plants (shown by arrows) after transformation with
pMON 73696. These dicamba tolerant plants were found to contain one
or more copies of DMO nucleotide as ascertained by the Invader.TM.
test. The example demonstrated the utility of DMO gene in producing
dicamba tolerant plants of other plant species.
All of the compositions and/or methods disclosed and claimed herein
can be made and executed without undue experimentation in light of
the present disclosure. While the compositions and methods of this
invention have been described in terms of preferred embodiments, it
will be apparent to those of skill in the art that variations may
be applied to the compositions and/or methods and in the steps or
in the sequence of steps of the method described herein without
departing from the concept, spirit and scope of the invention. More
specifically, it will be apparent that certain agents that are both
chemically and physiologically related may be substituted for the
agents described herein while the same or similar results would be
achieved. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the invention as defined by the appended
claims.
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Appln. WO 95/24492 PCT Appln. WO 97/31115 PCT Appln. WO 97/41228
Rojiyaa et al., 1987 (JP 1987201527-A), GenBank Accession E01312.
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2.sup.nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring
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98:503, 1975. Stalker et al., Science, 242:419, 1988. Streber and
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87:4144-4148, 1990.
SEQUENCE LISTINGS
1
2911023DNAArtificialBased on dicamba monooxygenase gene from
Pseudomonas maltophilia 1atggccactt tcgttagaaa cgcttggtac
gttgctgcac ttcctgagga gttgagcgag 60aagcctctag gaagaactat cctcgatact
ccactagctc tctatcgtca acctgacgga 120gttgtcgctg ccctgcttga
tatttgtccg catcgcttcg ctccgttgag tgacggtatt 180ctagtcaacg
gacatctcca gtgtccatat cacggtctgg aatttgacgg aggtggccag
240tgtgtccaca acccgcacgg caacggagcc cgccctgctt ctctgaacgt
gcgatcattc 300cctgtcgtgg aaagagacgc attgatctgg atctgccctg
gagatccagc actcgcagat 360cccggtgcta tccctgactt tgggtgtcgt
gttgatccag cttaccgtac tgtcggaggt 420tacggtcacg tggactgcaa
ctacaagctc cttgtggata acctcatgga tcttggacac 480gctcagtacg
tgcaccgcgc taacgcccaa acagacgcct tcgatagact tgagcgtgag
540gtgatcgttg gcgacggcga gatccaggcg ctcatgaaga tccctggtgg
cacaccctca 600gttctcatgg ctaagttctt gcgtggtgct aacacaccag
ttgacgcctg gaacgacatc 660cggtggaata aggtgtcggc tatgctgaac
ttcatcgcgg tcgcgccgga agggacgccg 720aaggagcagt caatccactc
ccgaggaacc catatcctta ctcctgagac cgaggcaagc 780tgccattact
tcttcggtag ttcccgcaac ttcggtatag acgatccaga gatggacggt
840gttctcagga gctggcaagc tcaagccctg gtgaaggagg acaaagtggt
cgttgaagct 900atcgaaaggc ggagggctta cgtcgaagcg aacgggatca
gacccgccat gttgtcctgc 960gacgaggcag ccgtcagggt atccagggag
attgagaagc tcgaacaact agaagcggcg 1020tga 10232340PRTArtificialBased
on dicamba monooxygenase gene from Pseudomonas maltophilia 2Met Ala
Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu 1 5 10 15
Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu 20
25 30 Ala Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp
Ile 35 40 45 Cys Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu
Val Asn Gly 50 55 60 His Leu Gln Cys Pro Tyr His Gly Leu Glu Phe
Asp Gly Gly Gly Gln 65 70 75 80 Cys Val His Asn Pro His Gly Asn Gly
Ala Arg Pro Ala Ser Leu Asn 85 90 95 Val Arg Ser Phe Pro Val Val
Glu Arg Asp Ala Leu Ile Trp Ile Cys 100 105 110 Pro Gly Asp Pro Ala
Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly 115 120 125 Cys Arg Val
Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val 130 135 140 Asp
Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His 145 150
155 160 Ala Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp
Arg 165 170 175 Leu Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln
Ala Leu Met 180 185 190 Lys Ile Pro Gly Gly Thr Pro Ser Val Leu Met
Ala Lys Phe Leu Arg 195 200 205 Gly Ala Asn Thr Pro Val Asp Ala Trp
Asn Asp Ile Arg Trp Asn Lys 210 215 220 Val Ser Ala Met Leu Asn Phe
Ile Ala Val Ala Pro Glu Gly Thr Pro 225 230 235 240 Lys Glu Gln Ser
Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu 245 250 255 Thr Glu
Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly 260 265 270
Ile Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln 275
280 285 Ala Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg
Arg 290 295 300 Arg Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met
Leu Ser Cys 305 310 315 320 Asp Glu Ala Ala Val Arg Val Ser Arg Glu
Ile Glu Lys Leu Glu Gln 325 330 335 Leu Glu Ala Ala 340
31023DNAArtificialBased on dicamba monooxygenase gene from
Pseudomonas maltophilia 3atgctcactt tcgttagaaa cgcttggtac
gttgctgcac ttcctgagga gttgagcgag 60aagcctctag gaagaactat cctcgatact
ccactagctc tctatcgtca acctgacgga 120gttgtcgctg ccctgcttga
tatttgtccg catcgcttcg ctccgttgag tgacggtatt 180ctagtcaacg
gacatctcca gtgtccatat cacggtctgg aatttgacgg aggtggccag
240tgtgtccaca acccgcacgg caacggagcc cgccctgctt ctctgaacgt
gcgatcattc 300cctgtcgtgg aaagagacgc attgatctgg atctgccctg
gagatccagc actcgcagat 360cccggtgcta tccctgactt tgggtgtcgt
gttgatccag cttaccgtac tgtcggaggt 420tacggtcacg tggactgcaa
ctacaagctc cttgtggata acctcatgga tcttggacac 480gctcagtacg
tgcaccgcgc taacgcccaa acagacgcct tcgatagact tgagcgtgag
540gtgatcgttg gcgacggcga gatccaggcg ctcatgaaga tccctggtgg
cacaccctca 600gttctcatgg ctaagttctt gcgtggtgct aacacaccag
ttgacgcctg gaacgacatc 660cggtggaata aggtgtcggc tatgctgaac
ttcatcgcgg tcgcgccgga agggacgccg 720aaggagcagt caatccactc
ccgaggaacc catatcctta ctcctgagac cgaggcaagc 780tgccattact
tcttcggtag ttcccgcaac ttcggtatag acgatccaga gatggacggt
840gttctcagga gctggcaagc tcaagccctg gtgaaggagg acaaagtggt
cgttgaagct 900atcgaaaggc ggagggctta cgtcgaagcg aacgggatca
gacccgccat gttgtcctgc 960gacgaggcag ccgtcagggt atccagggag
attgagaagc tcgaacaact agaagcggcg 1020tga 10234340PRTArtificialBased
on dicamba monooxygenase gene from Pseudomonas maltophilia 4Met Leu
Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu 1 5 10 15
Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu 20
25 30 Ala Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp
Ile 35 40 45 Cys Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu
Val Asn Gly 50 55 60 His Leu Gln Cys Pro Tyr His Gly Leu Glu Phe
Asp Gly Gly Gly Gln 65 70 75 80 Cys Val His Asn Pro His Gly Asn Gly
Ala Arg Pro Ala Ser Leu Asn 85 90 95 Val Arg Ser Phe Pro Val Val
Glu Arg Asp Ala Leu Ile Trp Ile Cys 100 105 110 Pro Gly Asp Pro Ala
Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly 115 120 125 Cys Arg Val
Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val 130 135 140 Asp
Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His 145 150
155 160 Ala Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp
Arg 165 170 175 Leu Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln
Ala Leu Met 180 185 190 Lys Ile Pro Gly Gly Thr Pro Ser Val Leu Met
Ala Lys Phe Leu Arg 195 200 205 Gly Ala Asn Thr Pro Val Asp Ala Trp
Asn Asp Ile Arg Trp Asn Lys 210 215 220 Val Ser Ala Met Leu Asn Phe
Ile Ala Val Ala Pro Glu Gly Thr Pro 225 230 235 240 Lys Glu Gln Ser
Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu 245 250 255 Thr Glu
Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly 260 265 270
Ile Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln 275
280 285 Ala Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg
Arg 290 295 300 Arg Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met
Leu Ser Cys 305 310 315 320 Asp Glu Ala Ala Val Arg Val Ser Arg Glu
Ile Glu Lys Leu Glu Gln 325 330 335 Leu Glu Ala Ala 340
51023DNAArtificialBased on dicamba monooxygenase gene from
Pseudomonas maltophilia 5atgctcactt tcgttagaaa cgcttggtac
gttgctgcac ttcctgagga gttgagcgag 60aagcctctag gaagaactat cctcgatact
ccactagctc tctatcgtca acctgacgga 120gttgtcgctg ccctgcttga
tatttgtccg catcgcttcg ctccgttgag tgacggtatt 180ctagtcaacg
gacatctcca gtgtccatat cacggtctgg aatttgacgg aggtggccag
240tgtgtccaca acccgcacgg caacggagcc cgccctgctt ctctgaacgt
gcgatcattc 300cctgtcgtgg aaagagacgc attgatctgg atctggcctg
gagatccagc actcgcagat 360cccggtgcta tccctgactt tgggtgtcgt
gttgatccag cttaccgtac tgtcggaggt 420tacggtcacg tggactgcaa
ctacaagctc cttgtggata acctcatgga tcttggacac 480gctcagtacg
tgcaccgcgc taacgcccaa acagacgcct tcgatagact tgagcgtgag
540gtgatcgttg gcgacggcga gatccaggcg ctcatgaaga tccctggtgg
cacaccctca 600gttctcatgg ctaagttctt gcgtggtgct aacacaccag
ttgacgcctg gaacgacatc 660cggtggaata aggtgtcggc tatgctgaac
ttcatcgcgg tcgcgccgga agggacgccg 720aaggagcagt caatccactc
ccgaggaacc catatcctta ctcctgagac cgaggcaagc 780tgccattact
tcttcggtag ttcccgcaac ttcggtatag acgatccaga gatggacggt
840gttctcagga gctggcaagc tcaagccctg gtgaaggagg acaaagtggt
cgttgaagct 900atcgaaaggc ggagggctta cgtcgaagcg aacgggatca
gacccgccat gttgtcctgc 960gacgaggcag ccgtcagggt atccagggag
attgagaagc tcgaacaact agaagcggcg 1020tga 10236340PRTArtificialBased
on dicamba monooxygenase gene from Pseudomonas maltophilia 6Met Leu
Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu 1 5 10 15
Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu 20
25 30 Ala Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp
Ile 35 40 45 Cys Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu
Val Asn Gly 50 55 60 His Leu Gln Cys Pro Tyr His Gly Leu Glu Phe
Asp Gly Gly Gly Gln 65 70 75 80 Cys Val His Asn Pro His Gly Asn Gly
Ala Arg Pro Ala Ser Leu Asn 85 90 95 Val Arg Ser Phe Pro Val Val
Glu Arg Asp Ala Leu Ile Trp Ile Trp 100 105 110 Pro Gly Asp Pro Ala
Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly 115 120 125 Cys Arg Val
Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val 130 135 140 Asp
Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His 145 150
155 160 Ala Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp
Arg 165 170 175 Leu Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln
Ala Leu Met 180 185 190 Lys Ile Pro Gly Gly Thr Pro Ser Val Leu Met
Ala Lys Phe Leu Arg 195 200 205 Gly Ala Asn Thr Pro Val Asp Ala Trp
Asn Asp Ile Arg Trp Asn Lys 210 215 220 Val Ser Ala Met Leu Asn Phe
Ile Ala Val Ala Pro Glu Gly Thr Pro 225 230 235 240 Lys Glu Gln Ser
Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu 245 250 255 Thr Glu
Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly 260 265 270
Ile Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln 275
280 285 Ala Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg
Arg 290 295 300 Arg Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met
Leu Ser Cys 305 310 315 320 Asp Glu Ala Ala Val Arg Val Ser Arg Glu
Ile Glu Lys Leu Glu Gln 325 330 335 Leu Glu Ala Ala 340
71023DNAArtificialBased on dicamba monooxygenase gene from
Pseudomonas maltophilia 7atggccacct tcgtccgcaa tgcctggtat
gtggcggcgc tgcccgagga actgtccgaa 60aagccgctcg gccggacgat tctcgacaca
ccgctcgcgc tctaccgcca gcccgacggt 120gtggtcgcgg cgctgctcga
catctgtccg caccgcttcg cgccgctgag cgacggcatc 180ctcgtcaacg
gccatctcca atgcccctat cacgggctgg aattcgatgg cggcgggcag
240tgcgtccata acccgcacgg caatggcgcc cgcccggctt cgctcaacgt
ccgctccttc 300ccggtggtgg agcgcgacgc gctgatctgg atctgtcccg
gcgatccggc gctggccgat 360cctggggcga tccccgactt cggctgccgc
gtcgatcccg cctatcggac cgtcggcggc 420tatgggcatg tcgactgcaa
ctacaagctg ctggtcgaca acctgatgga cctcggccac 480gcccaatatg
tccatcgcgc caacgcccag accgacgcct tcgaccggct ggagcgcgag
540gtgatcgtcg gcgacggtga gatacaggcg ctgatgaaga ttcccggcgg
cacgccgagc 600gtgctgatgg ccaagttcct gcgcggcgcc aatacccccg
tcgacgcttg gaacgacatc 660cgctggaaca aggtgagcgc gatgctcaac
ttcatcgcgg tggcgccgga aggcaccccg 720aaggagcaga gcatccactc
gcgcggtacc catatcctga cccccgagac ggaggcgagc 780tgccattatt
tcttcggctc ctcgcgcaat ttcggcatcg acgatccgga gatggacggc
840gtgctgcgca gctggcaggc tcaggcgctg gtcaaggagg acaaggtcgt
cgtcgaggcg 900atcgagcgcc gccgcgccta tgtcgaggcg aatggcatcc
gcccggcgat gctgtcgtgc 960gacgaagccg cagtccgtgt cagccgcgag
atcgagaagc ttgagcagct cgaagccgcc 1020tga 10238340PRTArtificialBased
on dicamba monooxygenase gene from Pseudomonas maltophilia 8Met Ala
Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu 1 5 10 15
Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu 20
25 30 Ala Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp
Ile 35 40 45 Cys Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu
Val Asn Gly 50 55 60 His Leu Gln Cys Pro Tyr His Gly Leu Glu Phe
Asp Gly Gly Gly Gln 65 70 75 80 Cys Val His Asn Pro His Gly Asn Gly
Ala Arg Pro Ala Ser Leu Asn 85 90 95 Val Arg Ser Phe Pro Val Val
Glu Arg Asp Ala Leu Ile Trp Ile Cys 100 105 110 Pro Gly Asp Pro Ala
Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly 115 120 125 Cys Arg Val
Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val 130 135 140 Asp
Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His 145 150
155 160 Ala Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp
Arg 165 170 175 Leu Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln
Ala Leu Met 180 185 190 Lys Ile Pro Gly Gly Thr Pro Ser Val Leu Met
Ala Lys Phe Leu Arg 195 200 205 Gly Ala Asn Thr Pro Val Asp Ala Trp
Asn Asp Ile Arg Trp Asn Lys 210 215 220 Val Ser Ala Met Leu Asn Phe
Ile Ala Val Ala Pro Glu Gly Thr Pro 225 230 235 240 Lys Glu Gln Ser
Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu 245 250 255 Thr Glu
Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly 260 265 270
Ile Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln 275
280 285 Ala Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg
Arg 290 295 300 Arg Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met
Leu Ser Cys 305 310 315 320 Asp Glu Ala Ala Val Arg Val Ser Arg Glu
Ile Glu Lys Leu Glu Gln 325 330 335 Leu Glu Ala Ala 340
91023DNAArtificialBased on dicamba monooxygenase gene from
Pseudomonas maltophilia 9atggccactt tcgttagaaa cgcttggtac
gttgctgcac ttcctgagga gttgagcgag 60aagcctctag gaagaactat cctcgatact
ccactagctc tctatcgtca acctgacgga 120gttgtcgctg ccctgcttga
tatttgtccg catcgcttcg ctccgttgag tgacggtatt 180ctagtcaacg
gacatctcca gtgtccatat cacggtctgg aatttgacgg aggtggccag
240tgtgtccaca acccgcacgg caacggagcc cgccctgctt ctctgaacgt
gcgatcattc 300cctgtcgtgg aaagagacgc attgatctgg atctggcctg
gagatccagc actcgcagat 360cccggtgcta tccctgactt tgggtgtcgt
gttgatccag cttaccgtac tgtcggaggt 420tacggtcacg tggactgcaa
ctacaagctc cttgtggata acctcatgga tcttggacac 480gctcagtacg
tgcaccgcgc taacgcccaa acagacgcct tcgatagact tgagcgtgag
540gtgatcgttg gcgacggcga gatccaggcg ctcatgaaga tccctggtgg
cacaccctca 600gttctcatgg ctaagttctt gcgtggtgct aacacaccag
ttgacgcctg gaacgacatc 660cggtggaata aggtgtcggc tatgctgaac
ttcatcgcgg tcgcgccgga agggacgccg 720aaggagcagt caatccactc
ccgaggaacc catatcctta ctcctgagac cgaggcaagc 780tgccattact
tcttcggtag ttcccgcaac ttcggtatag acgatccaga gatggacggt
840gttctcagga gctggcaagc tcaagccctg gtgaaggagg acaaagtggt
cgttgaagct 900atcgaaaggc ggagggctta cgtcgaagcg aacgggatca
gacccgccat gttgtcctgc 960gacgaggcag ccgtcagggt atccagggag
attgagaagc tcgaacaact agaagcggcg 1020tga
102310340PRTArtificialBased on dicamba monooxygenase gene from
Pseudomonas maltophilia 10Met Ala Thr Phe
Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu 1 5 10 15 Glu Leu
Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu 20 25 30
Ala Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp Ile 35
40 45 Cys Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu Val Asn
Gly 50 55 60 His Leu Gln Cys Pro Tyr His Gly Leu Glu Phe Asp Gly
Gly Gly Gln 65 70 75 80 Cys Val His Asn Pro His Gly Asn Gly Ala Arg
Pro Ala Ser Leu Asn 85 90 95 Val Arg Ser Phe Pro Val Val Glu Arg
Asp Ala Leu Ile Trp Ile Trp 100 105 110 Pro Gly Asp Pro Ala Leu Ala
Asp Pro Gly Ala Ile Pro Asp Phe Gly 115 120 125 Cys Arg Val Asp Pro
Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val 130 135 140 Asp Cys Asn
Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His 145 150 155 160
Ala Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp Arg 165
170 175 Leu Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln Ala Leu
Met 180 185 190 Lys Ile Pro Gly Gly Thr Pro Ser Val Leu Met Ala Lys
Phe Leu Arg 195 200 205 Gly Ala Asn Thr Pro Val Asp Ala Trp Asn Asp
Ile Arg Trp Asn Lys 210 215 220 Val Ser Ala Met Leu Asn Phe Ile Ala
Val Ala Pro Glu Gly Thr Pro 225 230 235 240 Lys Glu Gln Ser Ile His
Ser Arg Gly Thr His Ile Leu Thr Pro Glu 245 250 255 Thr Glu Ala Ser
Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly 260 265 270 Ile Asp
Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln 275 280 285
Ala Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg Arg 290
295 300 Arg Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met Leu Ser
Cys 305 310 315 320 Asp Glu Ala Ala Val Arg Val Ser Arg Glu Ile Glu
Lys Leu Glu Gln 325 330 335 Leu Glu Ala Ala 340
111020DNAPseudomonas maltophilia 11atgaccttcg tccgcaatgc ctggtatgtg
gcggcgctgc ccgaggaact gtccgaaaag 60ccgctcggcc ggacgattct cgacacaccg
ctcgcgctct accgccagcc cgacggtgtg 120gtcgcggcgc tgctcgacat
ctgtccgcac cgcttcgcgc cgctgagcga cggcatcctc 180gtcaacggcc
atctccaatg cccctatcac gggctggaat tcgatggcgg cgggcagtgc
240gtccataacc cgcacggcaa tggcgcccgc ccggcttcgc tcaacgtccg
ctccttcccg 300gtggtggagc gcgacgcgct gatctggatc tggcccggcg
atccggcgct ggccgatcct 360ggggcgatcc ccgacttcgg ctgccgcgtc
gatcccgcct atcggaccgt cggcggctat 420gggcatgtcg actgcaacta
caagctgctg gtcgacaacc tgatggacct cggccacgcc 480caatatgtcc
atcgcgccaa cgcccagacc gacgccttcg accggctgga gcgcgaggtg
540atcgtcggcg acggtgagat acaggcgctg atgaagattc ccggcggcac
gccgagcgtg 600ctgatggcca agttcctgcg cggcgccaat acccccgtcg
acgcttggaa cgacatccgc 660tggaacaagg tgagcgcgat gctcaacttc
atcgcggtgg cgccggaagg caccccgaag 720gagcagagca tccactcgcg
cggtacccat atcctgaccc ccgagacgga ggcgagctgc 780cattatttct
tcggctcctc gcgcaatttc ggcatcgacg atccggagat ggacggcgtg
840ctgcgcagct ggcaggctca ggcgctggtc aaggaggaca aggtcgtcgt
cgaggcgatc 900gagcgccgcc gcgcctatgt cgaggcgaat ggcatccgcc
cggcgatgct gtcgtgcgac 960gaagccgcag tccgtgtcag ccgcgagatc
gagaagcttg agcagctcga agccgcctga 102012339PRTPseudomonas
maltophilia 12Met Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu
Pro Glu Glu 1 5 10 15 Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu
Asp Thr Pro Leu Ala 20 25 30 Leu Tyr Arg Gln Pro Asp Gly Val Val
Ala Ala Leu Leu Asp Ile Cys 35 40 45 Pro His Arg Phe Ala Pro Leu
Ser Asp Gly Ile Leu Val Asn Gly His 50 55 60 Leu Gln Cys Pro Tyr
His Gly Leu Glu Phe Asp Gly Gly Gly Gln Cys 65 70 75 80 Val His Asn
Pro His Gly Asn Gly Ala Arg Pro Ala Ser Leu Asn Val 85 90 95 Arg
Ser Phe Pro Val Val Glu Arg Asp Ala Leu Ile Trp Ile Trp Pro 100 105
110 Gly Asp Pro Ala Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly Cys
115 120 125 Arg Val Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His
Val Asp 130 135 140 Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp
Leu Gly His Ala 145 150 155 160 Gln Tyr Val His Arg Ala Asn Ala Gln
Thr Asp Ala Phe Asp Arg Leu 165 170 175 Glu Arg Glu Val Ile Val Gly
Asp Gly Glu Ile Gln Ala Leu Met Lys 180 185 190 Ile Pro Gly Gly Thr
Pro Ser Val Leu Met Ala Lys Phe Leu Arg Gly 195 200 205 Ala Asn Thr
Pro Val Asp Ala Trp Asn Asp Ile Arg Trp Asn Lys Val 210 215 220 Ser
Ala Met Leu Asn Phe Ile Ala Val Ala Pro Glu Gly Thr Pro Lys 225 230
235 240 Glu Gln Ser Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu
Thr 245 250 255 Glu Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn
Phe Gly Ile 260 265 270 Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser
Trp Gln Ala Gln Ala 275 280 285 Leu Val Lys Glu Asp Lys Val Val Val
Glu Ala Ile Glu Arg Arg Arg 290 295 300 Ala Tyr Val Glu Ala Asn Gly
Ile Arg Pro Ala Met Leu Ser Cys Asp 305 310 315 320 Glu Ala Ala Val
Arg Val Ser Arg Glu Ile Glu Lys Leu Glu Gln Leu 325 330 335 Glu Ala
Ala 1329DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 13acggacgcgg agatgctcaa cttcatcgc
291424DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 14tccgctggaa caaggtgagc gcgt 241521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
15gtcgctgccc tgcttgatat t 211618DNAArtificial SequenceDescription
of Artificial Sequence Synthetic Primer 16cgccgcttct agttgttc
181757PRTPisum sativum 17Met Ala Ser Met Ile Ser Ser Ser Ala Val
Thr Thr Val Ser Arg Ala 1 5 10 15 Ser Arg Gly Gln Ser Ala Ala Met
Ala Pro Phe Gly Gly Leu Lys Ser 20 25 30 Met Thr Gly Phe Pro Val
Arg Lys Val Asn Thr Asp Ile Thr Ser Ile 35 40 45 Thr Ser Asn Gly
Gly Arg Val Lys Cys 50 55 1885PRTArabidopsis thaliana 18Met Ala Ser
Ser Met Leu Ser Ser Ala Thr Met Val Ala Ser Pro Ala 1 5 10 15 Gln
Ala Thr Met Val Ala Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala 20 25
30 Phe Pro Ala Thr Arg Lys Ala Asn Asn Asp Ile Thr Ser Ile Thr Ser
35 40 45 Asn Gly Gly Arg Val Asn Cys Met Gln Val Trp Pro Pro Ile
Glu Lys 50 55 60 Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Asp Leu
Thr Asp Ser Gly 65 70 75 80 Gly Arg Val Asn Cys 85
1976PRTArabidopsis thaliana 19Met Ala Gln Val Ser Arg Ile Cys Asn
Gly Val Gln Asn Pro Ser Leu 1 5 10 15 Ile Ser Asn Leu Ser Lys Ser
Ser Gln Arg Lys Ser Pro Leu Ser Val 20 25 30 Ser Leu Lys Thr Gln
Gln His Pro Arg Ala Tyr Pro Ile Ser Ser Ser 35 40 45 Trp Gly Leu
Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu Leu Arg 50 55 60 Pro
Leu Lys Val Met Ser Ser Val Ser Thr Ala Cys 65 70 75
2076PRTArabidopsis thaliana 20Met Ala Gln Val Ser Arg Ile Cys Asn
Gly Val Gln Asn Pro Ser Leu 1 5 10 15 Ile Ser Asn Leu Ser Lys Ser
Ser Gln Arg Lys Ser Pro Leu Ser Val 20 25 30 Ser Leu Lys Thr Gln
Gln His Pro Arg Ala Tyr Pro Ile Ser Ser Ser 35 40 45 Trp Gly Leu
Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu Leu Arg 50 55 60 Pro
Leu Lys Val Met Ser Ser Val Ser Thr Ala Cys 65 70 75 2172PRTPetunia
hybrida 21Met Ala Gln Ile Asn Asn Met Ala Gln Gly Ile Gln Thr Leu
Asn Pro 1 5 10 15 Asn Ser Asn Phe His Lys Pro Gln Val Pro Lys Ser
Ser Ser Phe Leu 20 25 30 Val Phe Gly Ser Lys Lys Leu Lys Asn Ser
Ala Asn Ser Met Leu Val 35 40 45 Leu Lys Lys Asp Ser Ile Phe Met
Gln Lys Phe Cys Ser Phe Arg Ile 50 55 60 Ser Ala Ser Val Ala Thr
Ala Cys 65 70 2269PRTTriticum aestivum 22Met Ala Ala Leu Val Thr
Ser Gln Leu Ala Thr Ser Gly Thr Val Leu 1 5 10 15 Ser Val Thr Asp
Arg Phe Arg Arg Pro Gly Phe Gln Gly Leu Arg Pro 20 25 30 Arg Asn
Pro Ala Asp Ala Ala Leu Gly Met Arg Thr Val Gly Ala Ser 35 40 45
Ala Ala Pro Lys Gln Ser Arg Lys Pro His Arg Phe Asp Arg Arg Cys 50
55 60 Leu Ser Met Val Val 65 23171DNAPisum sativum 23atggcttcta
tgatatcctc ttccgctgtg acaacagtca gccgtgcctc tagggggcaa 60tccgccgcaa
tggctccatt cggcggcctc aaatccatga ctggattccc agtgaggaag
120gtcaacactg acattacttc cattacaagc aatggtggaa gagtaaagtg c
17124255DNAArabidopsis thaliana 24atggcttcct ctatgctctc ttccgctact
atggttgcct ctccggctca ggccactatg 60gtcgctcctt tcaacggact taagtcctcc
gctgccttcc cagccacccg caaggctaac 120aacgacatta cttccatcac
aagcaacggc ggaagagtta actgtatgca ggtgtggcct 180ccgattgaaa
agaagaagtt tgagactctc tcttaccttc ctgaccttac cgattccggt
240ggtcgcgtca actgc 25525228DNAArabidopsis thaliana 25atggcgcaag
ttagcagaat ctgcaatggt gtgcagaacc catctcttat ctccaatctc 60tcgaaatcca
gtcaacgcaa atctccctta tcggtttctc tgaagacgca gcagcatcca
120cgagcttatc cgatttcgtc gtcgtgggga ttgaagaaga gtgggatgac
gttaattggc 180tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca cggcgtgc
22826228DNAArtificial sequenceDescription of Artificial Sequence
Artificial primer 26atggcgcaag ttagcagaat ctgcaatggt gtgcagaacc
catctcttat ctccaatctc 60tcgaaatcca gtcaacgcaa atctccctta tcggtttctc
tgaagacgca gcagcatcca 120cgagcttatc cgatttcgtc gtcgtgggga
ttgaagaaga gtgggatgac gttaattggc 180tctgagcttc gtcctcttaa
ggtcatgtct tctgtttcca cggcgtgc 22827216DNAArtificial
sequenceDescription of Artificial Sequence Artificial primer
27atggcccaga tcaacaacat ggcccagggc atccagaccc tgaaccctaa ctctaacttc
60cacaagccgc aagtgcccaa gtctagctcc ttcctcgtgt tcggctccaa gaagctcaag
120aatagcgcca attccatgct ggtcctgaag aaagactcga tcttcatgca
gaagttctgc 180tcctttcgca tcagtgcttc ggttgcgact gcctgc
21628207DNAArtificial sequenceDescription of Artificial Sequence
Artificial primer 28atggcggcac tggtgacctc ccagctcgcg acaagcggca
ccgtcctgtc ggtgacggac 60cgcttccggc gtcccggctt ccagggactg aggccacgga
acccagccga tgccgctctc 120gggatgagga cggtgggcgc gtccgcggct
cccaagcaga gcaggaagcc acaccgtttc 180gaccgccggt gcttgagcat ggtcgtc
20729433DNAArtificialDescription of Artificial Sequence Artificial
primer 29agatcttgag ccaatcaaag aggagtgatg tagacctaaa gcaataatgg
agccatgacg 60taagggctta cgcccatacg aaataattaa aggctgatgt gacctgtcgg
tctctcagaa 120cctttacttt ttatgtttgg cgtgtatttt taaatttcca
cggcaatgac gatgtgaccc 180aacgagatct tgagccaatc aaagaggagt
gatgtagacc taaagcaata atggagccat 240gacgtaaggg cttacgccca
tacgaaataa ttaaaggctg atgtgacctg tcggtctctc 300agaaccttta
ctttttatat ttggcgtgta tttttaaatt tccacggcaa tgacgatgtg
360acctgtgcat ccgctttgcc tataaataag ttttagtttg tattgatcga
cacggtcgag 420aagacacggc cat 433
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