U.S. patent application number 11/439396 was filed with the patent office on 2006-09-21 for agrobacterium-mediated transformation of turfgrass.
This patent application is currently assigned to Rutgers, The State University Of New Jersey. Invention is credited to Subha R. Lakkaraju, Lynne H. Pitcher, Barbara A. Zilinskas.
Application Number | 20060212973 11/439396 |
Document ID | / |
Family ID | 36568890 |
Filed Date | 2006-09-21 |
United States Patent
Application |
20060212973 |
Kind Code |
A1 |
Zilinskas; Barbara A. ; et
al. |
September 21, 2006 |
Agrobacterium-mediated transformation of turfgrass
Abstract
A method of obtaining transgenic turfgrass plants by an
Agrobacterium-mediated transformation protocol is disclosed. The
protocol makes use of a modified Agrobacterium vector system in
which selectable marker genes and other genes of interest are
operably linked to strong promoters from monocotyledenous plants,
such as actin and ubiquitin promoters, that function efficiently in
turfgrass cells. Transgenic turfgrass plants of several species,
produced by the Agrobacterium-mediated transformation method, are
also disclosed.
Inventors: |
Zilinskas; Barbara A.;
(Princeton Junction, NY) ; Pitcher; Lynne H.;
(Highland Park, NJ) ; Lakkaraju; Subha R.; (East
Brunswick, NJ) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE, 46TH FLOOR
1650 MARKET STREET
PHILADELPHIA
PA
19103
US
|
Assignee: |
Rutgers, The State University Of
New Jersey
|
Family ID: |
36568890 |
Appl. No.: |
11/439396 |
Filed: |
May 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09743840 |
Jan 17, 2001 |
7057090 |
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PCT/US99/16001 |
Jul 15, 1999 |
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11439396 |
May 22, 2006 |
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60093163 |
Jul 17, 1998 |
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Current U.S.
Class: |
800/294 ;
800/320 |
Current CPC
Class: |
C12N 15/8205 20130101;
A01H 5/12 20130101 |
Class at
Publication: |
800/294 ;
800/320 |
International
Class: |
C12N 15/82 20060101
C12N015/82; A01H 5/00 20060101 A01H005/00; A01H 1/00 20060101
A01H001/00 |
Claims
1-21. (canceled)
22. A method of producing a transgenic turfgrass plant, comprising
the steps of: (a) culturing embryogenic tissue from seeds of a
turfgrass plant on a medium that promotes de-differentiation of the
tissue, to produce regenerable callus tissue; (b) inoculating the
callus tissue with Agrobacterium carrying at least one vector for
transformation, the vector comprising virulence genes from plasmid
pSB1 or pSB4, in which vector is inserted a heterologous DNA
construct and a selectable marker conferring antibiotic resistance
to transformed cells, wherein the DNA construct and selectable
marker are operably linked to a promoter from a monocotyledonous
species, wherein the inoculating comprises mixing the callus tissue
with the Agrobacterium under conditions permitting the
Agrobacterium to infiltrate the callus tissue, thereby forming
Agrobacterium-infiltrated callus tissue; (c) co-culturing the
Agrobacterium-infiltrated callus tissue under conditions that
enable the Agrobacterium vector to transform cells of the
Agrobacterium-infiltrated callus tissue; (d) selecting transformed
cells by culturing the Agrobacterium-infiltrated callus tissue on a
selection medium comprising an antibiotic, wherein the transformed
cells are resistant to the antibiotic and are selected by their
growth in the presence of the antibiotic; and (e) regenerating a
transformed turfgrass plant from the transformed cells.
23. The method of claim 1, wherein the turfgrass is a species
selected from the group consisting of creeping bentgrass, tall
fescue, velvet bentgrass, perennial ryegrass, hard fescue, Chewings
fescue, strong creeping fescue, colonial bentgrass and Kentucky
bluegrass.
24. The method of claim 1, wherein the promoter is selected from
the group consisting of maize ubiquitin gene promoters, rice actin
gene promoters, maize Adh 1 gene promoters, maize tubulin (Tub A, B
or C) gene promoters, and rice tubulin (Tub A, B or C) gene
promoters.
25. The method of claim 1, wherein the selectable marker gene
confers hygromycin resistance on transformed cells.
26. The method of claim 1, wherein the vector comprises a transgene
selected from the group consisting of: (a) a gene encoding glucose
oxidase; (b) a gene encoding citrate synthase; (c) a gene encoding
.DELTA.-9 desaturase from Saccharomyces cerevisiae or Cryptococcus
curvatus; (d) a gene encoding .DELTA.-11 desaturase; (e) a gene
encoding a plant homolog of the neutrophil NADPH oxidase; (f) a
gene encoding bacteriopsin from Halobacterium halobium; and (g) a
gene encoding pokeweed antiviral protein.
Description
[0001] This application claims priority to U.S. Provisional
Application No. 60/093,163, filed Jul. 17, 1998, the entirety of
which is incorporated by reference herein.
FIELD OF THE INVENTION
[0002] This invention relates to the field of plant transformation
methods. More specifically, an Agrobacterium-mediated method for
transforming turfgrass is provided, as well as transgenic turfgrass
produced by the method.
BACKGROUND OF THE INVENTION
[0003] Various scientific articles and patents are referred to in
parentheses throughout the specification. These documents are
incorporated by reference herein to describe the state of the art
to which this invention pertains. Full citations of the scientific
articles appear at the end of the specification.
[0004] Turfgrasses and turfgrass breeding are of significant
economic importance worldwide. In recent years, traditional
breeding programs have been augmented by molecular biological and
recombinant techniques. However, similar to most monocotyledenous
plants, turfgrasses have proven recalcitrant to tissue culture,
transformation and regeneration procedures. Among these,
Agrobacterium-mediated transformation of a turfgrass heretofore has
not been accomplished.
[0005] Turfgrass transformation has been achieved using direct
methods of DNA transfer, including protoplast transformation and
particle gun bombardment. Nonetheless, Agrobacterium-mediated
transformation offers several advantages over particle gun
bombardment or other means of direct gene transfer. These include
stable transgene integration without rearrangement of either host
or transgene DNA; preferential integration of the transgene into
transcriptionally active regions of the genome; ability to transfer
large pieces of DNA; and integration of low numbers of gene copies
into plant nuclear DNA which is particularly important to minimize
possible co-suppression of the transgene in later generations.
[0006] Until recently, Agrobacterium-mediated transformation was
thought to be limited to dicotyledonous plants. However, Hiei et
al. in 1994 described efficient transformation of rice by
Agrobacterium, and subsequently there have been convincing reports
for maize, barley and wheat (Ishida et al., 1996; Tingay et al.,
1997; Cheng et al., 1997; see also U.S. Pat. No. 5,591,616 to Hiei
et al). Numerous factors are of critical importance in
Agrobacterium-mediated transformation of monocots, including the
type and stage of tissue that is infected, the vector and bacterial
strains used, plant genotype, tissue culture conditions, and the
actual infection process. As a result, methods that have proven
successful for Agrobacterium-mediated transformation of some
monocots, such as rice and maize, have not been successful for
transforming turfgrass.
[0007] An object of the present invention is to develop an
efficient and reliable transformation system for turfgrass,
mediated by Agrobacterium tumefaciens. Another object of the
invention is to regenerate transgenic plants containing one or more
foreign genes introduced by Agrobacterium tumefaciens-mediated
transformation.
SUMMARY OF THE INVENTION
[0008] In accordance with the objects of the present invention, an
Agrobacterium-mediated transformation system for turfgrass is
provided, which is efficient and reliable. The invention also
provides for the development of transgenic plants containing one or
more transgenes of significant practical utility.
[0009] According to one aspect of the invention, a method of
producing a transgenic turfgrass plant is provided. The method
comprises: (a) providing regenerable callus tissue from the
turfgrass plant; (b) inoculating the tissue with Agrobacterium
carrying at least one vector for transformation, the vector
comprising virulence genes that confer strong infectivity to
Agrobacterium, in which vector is inserted a heterologous DNA
construct operably linked to a promoter from a monocotylednous
species, and a selectable marker gene conferring antibiotic
resistance to transformed cells operably linked to a promoter from
a monocotyledenous species; (c) culturing the inoculated tissue
under conditions that enable the Agrobacterium vector to transform
cells of the tissue; (d) selectively culturing the inoculated
tissue on a selection medium comprising the antibiotic; and (e)
regenerating a transformed turfgrass plant from the selectively
cultured tissue.
[0010] Preferably, the turfgrass is a species selected from the
group consisting of creeping bentgrass, tall fescue, velvet
bentgrass, perennial ryegrass, hard fescue, Chewings fescue, strong
creeping fescue, colonial bentgrass and Kentucky bluegrass. In
another preferred embodiment, the Agrobacterium comprises a binary
vector system and the virulence genes therein are obtained from a
plasmid within Agrobacterium tumefaciens strain 281. The promoter
is preferably selected from the group consisting of maize ubiquitin
gene promoters, rice actin gene promoters, maize Adh 1 gene
promoters, rice or maize tubulin (Tub A, B or C) gene promoters,
and alfalfa His 3 gene promoters. The selectable marker gene
preferably confers hygromycin resistance on transformed tissue. The
callus used for the transformation preferably is obtained by
culturing seeds of the turfgrass on a de-differentiation
medium.
[0011] Also provided in accordance with another aspect of the
invention is a transgenic turfgrass plant prepared by the
aforementioned method. Seeds of the transgenic plant are provided
as well. In preferred embodiments, the transgenic turfgrass plant
comprises a transgene selected from the group consisting of genes
encoding glucose oxidase, citrate synthase, -9 desaturase from
Saccharomyces cerevisiae or Cryptococcus curvatus, -11 desaturase,
a plant homolog of the neutrophil NADPH oxidase, a bacteriopsin
from Halobacterium halobium, or pokeweed antiviral protein.
[0012] According to another aspect of the invention, a superbinary
vector for Agrobacterium-mediated transformation of turfgrass is
provided. The vector comprises: (a) a virulence region from a Ti
plasmid of an A. tumefaciens strain that confers to the strain as
strong a virulence as that displayed by A. tumefaciens strain 281;
(b) a selectable marker gene operably linked to a promoter obtained
from a gene of a monocotyledenous plant; and (c) a site for
insertion of at least one additional coding sequence, operably
linked to a promoter obtained from a gene of a monocotyledenous
plant, the promoter being the same as or different from the
promoter operably linked to the selectable marker gene. In
preferred embodiments the virulence region is obtained from
Agrobacterium strain 281. The promoter is selected from the group
consisting of maize ubiquitin gene promoters, rice actin gene
promoters, maize Adh 1 gene promoters, rice or maize tubulin (Tub
A, B or C) gene promoters, and alfalfa His 3 gene promoters. The
selectable marker gene preferably confers hygromycin resistance on
transformed cells. The site for insertion of the additional coding
sequence preferably comprises a coding sequence of a reporter gene,
and/or comprises a coding sequence of a gene encoding the useful
proteins listed above.
[0013] According to another aspect of the invention, a turfgrass
plant cell, transformed with the aforementioned Agrobacterium
vector is provided. Preferably the turfgrass is creeping bentgrass,
tall fescue, velvet bentgrass, perennial ryegrass, hard fescue,
Chewings fescue, strong creeping fescue, colonial bentgrass or
Kentucky bluegrass. A transgenic turfgrass plant regenerated from
the aforementioned transformed cell is also provided, as are seeds
of the transgenic turfgrass plant.
[0014] Other features and advantages of the present invention will
be understood by reference to the detailed description and examples
that follow.
DETAILED DESCRIPTION OF THE INVENTION
I. DEFINITIONS
[0015] Various terms used throughout the specification and claims
to describe the invention. Unless otherwise specified, these terms
are defined as set forth below.
[0016] With reference to nucleic acid molecules, the term "isolated
nucleic acid" is sometimes used. This term, when applied to DNA,
refers to a DNA molecule that is separated from sequences with
which it is immediately contiguous (in the 5' and 3' directions) in
the naturally occurring genome of the organism from which it was
derived. For example, the "isolated nucleic acid" may comprise a
DNA molecule inserted into a vector, such as a plasmid or virus
vector, or integrated into the genomic DNA of a prokaryote or
eukaryote. An "isolated nucleic acid molecule" may also comprise a
cDNA molecule.
[0017] With respect to RNA molecules, the term "isolated nucleic
acid" primarily refers to an RNA molecule encoded by an isolated
DNA molecule as defined above. Alternatively, the term may refer to
an RNA molecule that has been sufficiently separated from RNA
molecules with which it would be associated in its natural state
(i.e., in cells or tissues), such that it exists in a
"substantially pure" form (the term "substantially pure" is defined
below).
[0018] The term "substantially pure" refers to a preparation
comprising at least 50-60% by weight the compound of interest (e.g.
nucleic acid, oligonucleotide, protein, etc.). More preferably, the
preparation comprises at least 75% by weight, and most preferably
90-99% by weight, the compound of interest. Purity is measured by
methods appropriate for the compound of interest (e.g.
chromatographic methods, agarose or polyacrylamide gel
electrophoresis, HPLC analysis, and the like).
[0019] When used herein in describing components of media or other
experimental results, the term "about" means within a margin of
commonly acceptable error for the determination being made, using
standard methods. For tissue culture media in particular, persons
skilled in the art would appreciate that the concentrations of
various components initially added to culture media may change
somewhat during use of the media, e.g., by evaporation of liquid
from the medium or by condensation onto the medium. Moreover, it is
understood that the precise concentrations of the macronutrients,
vitamins and carbon sources are less critical to the efficacy of
the media than are the micronutrient, hormone and antibiotic
concentrations.
[0020] Nucleic acid sequences and amino acid sequences can be
compared using computer programs that align the similar sequences
of the nucleic or amino acids thus define the differences. In the
comparisons made in the present invention, the CLUSTLW program and
parameters employed therein were utilized (Thompson et al., 1994,
supra). However, equivalent alignments and similarity/identity
assessments can be obtained through the use of any standard
alignment software. For instance, the BLAST programs used to query
sequence similarity in GenBank and other public databases may be
used. The GCG Wisconsin Package version 9.1, available from the
Genetics Computer Group in Madison, Wis., and the default
parameters used (gap creation penalty=12. gap extension penalty=4)
by that program may also be used to compare sequence identity and
similarity.
[0021] The term "substantially the same" refers to nucleic acid or
amino acid sequences having sequence variations that do not
materially affect the functionality of cis acting regulatory
sequences (e.g, promoters, transcriptional response elements, etc.)
or the nature of the encoded gene product (i.e. the structure,
stability characteristics, substrate specificity and/or biological
activity of the protein). With particular reference to nucleic acid
sequences, the term "substantially the same" is intended to refer
to conserved sequences governing expression and to the coding
region (referring primarily to degenerate codons encoding the same
amino acid; or alternate codons encoding conservative substitute
amino acids in the encoded polypeptide).
[0022] The terms "percent identical" and "percent similar" are also
used herein in comparisons among nucleic acid sequences. When
referring to nucleic acid molecules, "percent identical" refers to
the percent of the nucleotides of the subject nucleic acid sequence
that have been matched to identical nucleotides by a sequence
analysis program. When referring to amino acid sequences, "percent
identical" refers to the percent of the amino acids of the subject
amino acid sequence that have been matched to identical amino acids
in the compared amino acid sequence by a sequence analysis program.
"Percent similar" refers to the percent of the amino acids of the
subject amino acid sequence that have been matched to identical or
conserved amino acids. Conserved amino acids are those which differ
in structure but are similar in physical properties such that the
exchange of one for another would not appreciably change the
tertiary structure of the resulting protein. Conservative
substitutions are defined in Taylor (1986, J. Theor. Biol.
11I9:205).
[0023] With respect to oligonucleotides or other single-stranded
nucleic acid molecules, the term "specifically hybridizing" refers
to the association between two single-stranded nucleic acid
molecules of sufficiently complementary sequence to permit such
hybridization under pre-determined conditions generally used in the
art (sometimes termed "substantially-complementary"). In
particular, the term refers to hybridization of an oligonucleotide
with a substantially complementary sequence contained within a
single-stranded DNA or RNA molecule, to the substantial exclusion
of hybridization of the oligonucleotide with single-stranded
nucleic acids of non-complementary sequence.
[0024] A "coding sequence" or "coding region" refers to a nucleic
acid molecule having sequence information necessary to produce a
gene product, when the sequence is expressed.
[0025] The term "operably linked" or "operably inserted" means that
the regulatory sequences necessary for expression of the coding
sequence are placed in a nucleic acid molecule in the appropriate
positions relative to the coding sequence so as to enable
expression of the coding sequence. This same definition is
sometimes applied to the arrangement other transcription control
elements (e.g. enhancers) in an expression vector.
[0026] When describing the organization of a nucleic acid molecule,
the term "upstream" refers to the 5' direction and the term
"downstream" refers to the 3' direction.
[0027] The term "reporter gene" refers to a nucleic acid coding
sequence that encodes a readily detectable gene product, which may
be operably linked to a promoter region to form a chimeric gene,
such that expression of the coding sequence is regulated by the
promoter and the product of the coding sequence is readily
assayed.
[0028] The term "selectable marker gene" refers to a gene that when
expressed confers a selectable phenotype, such as antibiotic
resistance, on a transformed cell or plant.
[0029] Transcriptional and translational control sequences,
sometimes referred to herein as "expression control" sequences or
elements, or "expression regulating" sequences or elements, are DNA
regulatory elements such as promoters, enhancers, ribosome binding
sites, polyadenylation signals, terminators and the like, that
provide for the expression of a coding sequence in a host cell. The
term "expression" is intended to include transcription of DNA and
translation of the mRNA transcript.
[0030] The terms "promoter", "promoter region" or "promoter
sequence" refer generally to transcriptional regulatory regions of
a gene, which may be found at the 5' or 3' side of the coding
region, or within the coding region, or within introns. Typically,
a promoter is a DNA regulatory region capable of binding RNA
polymerase in a cell and initiating transcription of a downstream
(3' direction) coding sequence. The typical 5' promoter sequence is
bounded at its 3' terminus by the transcription initiation site and
extends upstream (5' direction) to include the minimum number of
bases or elements necessary to initiate transcription at levels
detectable above background. Within the promoter sequence is a
transcription initiation site (conveniently defined by mapping with
nuclease S1), as well as protein binding domains (consensus
sequences) responsible for the binding of RNA polymerase.
[0031] A "vector" is a replicon, such as plasmid, phage, cosmid, or
virus to which another nucleic acid segment may be operably
inserted so as to bring about the replication or expression of the
segment.
[0032] The term "nucleic acid construct" or "DNA construct" refers
to genetic sequence used to transform plant cells and generate
progeny transgenic plants. At minimum a DNA construct comprises a
coding region for a selected gene product, operably linked to 5'
and 3' regulatory sequences for expression in transformed plants.
In preferred embodiments, such constructs are chimeric, i.e., the
coding sequence is from a different source one or more of the
regulatory sequences (e.g., coding sequence from tobacco and
promoter from maize). However, non-chimeric DNA constructs also can
be used. In addition to methods specifically described herein, the
transforming DNA may be prepared according to standard protocols
such as those set forth in Ausubel et al. (1999). A plant species
or cultivar may be transformed with a DNA construct (chimeric or
non-chimeric) that encodes a polypeptide from a different plant
species, or cultivar, or a non-plant species. Alternatively, a
plant species or cultivar may be transformed with a DNA construct
(chimeric or non-chimeric) that encodes a polypeptide from the same
plant species or cultivar. The term "transgene" is sometimes used
to refer to the DNA construct within the transformed cell or
plant.
[0033] A cell has been "transformed" or "transfected" by a DNA
construct when such DNA has been introduced inside the cell. The
transforming DNA may or may not be integrated (covalently linked)
into the genome of the cell. For example, the transforming DNA may
be maintained on an episomal element such as a plasmid. With
respect to eukaryotic cells, a stably transformed cell is one in
which the transforming DNA has become integrated into a chromosome
so that it is inherited by daughter cells through chromosome
replication. This stability is demonstrated by the ability of the
eukaryotic cell to establish cell lines or clones comprised of a
population of daughter cells containing the transforming DNA. A
"clone" is a population of cells derived from a single cell or
common ancestor by mitosis. A "cell line" is a clone of a primary
cell that is capable of stable growth in vitro for many
generations.
II. DESCRIPTION
[0034] The present invention provides an efficient and reliable
transformation system for turfgrass species. The species chosen to
develop this system uses creeping bentgrass, Agrostis palustris
Huds. Due to the reasonably close similarity among various
turfgrasses (i.e., in physiology, genome organization, etc.), this
transformation system will have broad applicability to many
different kinds of turfgrass. For instance, the system also has
been applied to velvet bentgrass, Agrostis canina L. and tall
fescue, Festuca arundinacea Scheb., and although the transformation
efficiency is not as great as for creeping bentgrass, it is clear
that transformants can be obtained from several species. Other
turfgrasses contemplated for the transformation system of the
invention include, but are not limited to, perennial ryegrass, hard
fescue, Chewings fescue, strong creeping fescue, colonial bentgrass
and Kentucky bluegrass.
[0035] Binary vectors are commonly used in Agrobacterium-mediated
transformation. Recently, a "superbinary" vector system was
developed (Komari et al., 1990; Saito et al., 1992; Hiei et al.,
1994; U.S. Pat. No. 5,731,139 to Komari et al.). In this system the
plasmid that carries the T-DNA also contains certain virulence
genes from strain A281, which is known for high efficiency of
transformation.
[0036] In arriving at the present invention, the conditions and
vectors described for transformation of rice (Hiei er al., 1994)
and maize (Ishida et al., 1996) were initially used, but without
success in creeping bentgrass. It was suspected that a major
problem with these vectors was the CaMV35S promoter which drives
the expression of the selectable marker in the rice and maize
systems. Therefore, a new vector was constructed, which has the
components of the "superbinary vector" developed by Hiei et al.
(1994), coupled with a suitable selectable marker (hygromycin
resistance) and the easily scorable-glucuronidase (GUS) reporter
gene. More significantly, in the new vector, the expression of each
of these respective transgenes is driven by promoters for strong,
constitutive expression in monocots, e.g., the rice actin and maize
ubiquitin promoters. Independent transformations, conducted over
the course of several months, demonstrated that an Agrobacterium
tumefaciens such as this can indeed transfer DNA into the
chromosomes of various turfgrasses.
[0037] Certain features of the Agrobacterium-mediated turfgrass
transformation system describe herein are believed to be
particularly important for successful transformation and
regeneration of transgenic turfgrass. One of these is the use of Ti
plasmids from strongly infective Agrobacterium strains. Preferred
for use are superbinary hybrid Agrobacterium vectors, such as pSB1
and pSB11 (Komari et al., 1996). These and other superbinary
vectors that can be modified for use in the present invention are
described in U.S. Pat. No. 5,731,179 to Komari et al. These vectors
contain a DNA region containing virB gene and virG gene in
virulence region of Ti plasmid pTiBo542 of Agrobacterium
tumefaciens, which is a Ti plasmid contained in A. tumefaciens
strain A281 (ATCC Accession No. 37349), a strain known for its
strong virulence. The virB and virG genes in the virulence region
of pTiBo542 are contained a 15.2 kb KpnI restriction fragment,
which itself may be used in the present invention. Moreover,
although vectors comprising the hypervirulence-conferring portions
of pTiBo542 are, exemplified herein, it will be appreciated by
persons skilled in the art that corresponding genes from any highly
virulent Agrobacterium strain may be used instead. These include
strains that are presently available as well as strains that may be
discovered in the future.
[0038] A particularly important feature of the invention is the
modification of Agrobacterium vectors to contain promoters and
other regulatory sequences particularly suited for expression in
turfgrasses. Thus, the strong constitutive promoters from the rice
actin gene and the maize ubiquitin gene are exemplified herein.
However, any constitutively expressed or inducible promoter for
expression in monocots may be used in the present invention.
Examples of other constitutive promoters suitable for use in the
present invention are the maize Adh 1 promoter, the rice or maize
tubulin (Tub A, B or C) promoters; and the alfalfa His 3 promoter.
Monocotyledenous tissue-specific promoters also may be utilized.
For example, seed-specific promoters suitable for use in the
present invention include zein promoters, such as the 27-kDa zein
promoter or the 10-kDa zein promoter.
[0039] The choice of selectable marker is also important.
Hygromycin is exemplified herein as a selectable marker. However,
other selectable markers are also suitable for use in the
invention. For instance, resistance to phosphothricin herbicides is
particularly useful for selecting transformed monocotyledenous
plant cells. In addition, kanamycin is routinely used as a
selection medium for plant transformation, though it is less
preferred for use in the present invention than is hygromycin.
[0040] For Agrobacterium-mediated transformation, an antibiotic to
eliminate Agrobacterium is included in the selection medium.
Cefotaxime is the preferred antibiotic for this purpose. However,
Augmentin (amoxicillin and lithium clavulenate) and carbenincillin
also have been found effective for eliminating Agrobacterium.
[0041] If a reporter gene is utilized, it can be any one of several
commonly used in the art. Examples of suitable reporter genes
include, but are not limited to, genes encoding-glucuronidase
(GUS), luciferase, chloramphenicol acetyl transferase (CAT), green
fluorescent protein (GFP) and modified forms of GFP (e.g., EGFP,
EPFP and GFP.sub.UV.
[0042] In addition, the use of friable, regenerable callus for the
transformation protocol is considered particularly important to
successful, efficient transformation and regeneration of intact
plants. In a preferred embodiment, the callus is generated from
embryogenic tissue, most preferably mature seeds, though it may
also be generated from seed parts or immature seeds. Other
organogenic tissues may also be utilized as starting material for
callus growth. It has been found in accordance with the present
invention that the culture media described herein will facilitate
the growth of appropriate (i.e. friable, regenerable) callus
tissue, which is best harvested as soon as a sufficient amount of
callus is generated from the starting material.
[0043] To optimize the conditions for efficient transformation of
turfgrass, a number of parameters may be systematically altered to
determine the best conditions to achieve efficient and easily
reproducible transformation. Conditions that may be altered
include: 1) the culture medium and induction agents immediately
prior to and during co-cultivation with Agrobacterium; 2)
inoculation and co-culture methods and time period; 3) the presence
and absence of surfactants in the inoculation medium; and 4) use of
embryogenic callus versus suspension cell cultures. Optimum
conditions have been established for creeping bentgrass.
Modifications of these conditions have enabled the transformation
of callus obtained from velvet bentgrass: and tall fescue.
[0044] Thus, the present invention provides a method for generating
transgenic turfgrass using Agrobacterium vectors, that heretofore
has been unavailable. A preferred embodiment of the present
invention comprises the following transformation/regeneration
protocol, based on Agrobacterium-mediated transformation.
[0045] 1) The starting tissue (e.g., mature seeds in a particularly
preferred embodiment) of the selected turfgrass are
surface-sterilized and placed on a standard de-differentiation
tissue culture medium (see the examples) for about 3-6 weeks,
preferably in the dark, at room temperature.
[0046] 2) Proliferating calli are selected and transferred to fresh
medium of the same type on a regular basis. Only callus that is
friable and regenerable should be selected for further culture.
[0047] 3) Prior to exposure to Agrobacterium, the chosen callus is
transferred to new medium to promote active cell division; the
callus is used for transformation within about a week of the final
transfer to fresh medium.
[0048] 4) The Agrobacterium strain carrying the transforming
plasmids is grown, induced with acetosyringone (a phenolic compound
demonstrated to induce Agrobacterium vir gene expression) and
resuspended in an inoculation medium comprising acetosyringone and,
optionally, a surfactant such as pluronic F-68 or another suitable
surfactant.
[0049] 5) Optionally, callus may be pre-treated prior to
inoculation with Agrobacterium by vacuum-infiltration with
inoculation medium containing acetosyringone.
[0050] 6) The callus tissue is then placed in the presence of the
Agrobacterium suspension to allow the bacteria to infiltrate the
callus tissue. Optionally, excess Agrobacterium may thereafter be
removed from the calli by gentle vacuum filtration. Calli, on a
sterile filter, are then placed on co-cultivation solid-medium
(standard de-differentiation medium but containing glucose and
acetosyringone). Co-cultivation of the calli with the Agrobacterium
cells that remain associated with the calli is allowed to proceed
for a few (e.g., three) days, the object being to allow sufficient
time for penetration of the T-DNA into the plant cells while
avoiding overgrowth of the calli with the Agrobacterium.
[0051] 7) The calli are then transferred to selection medium
containing the selection antibiotic (e.g., hygromycin) and the
antibiotic for removal of the Agrobacterium. Calli are kept on this
medium for several weeks (e.g. 6-8 weeks) and checked periodically
for proliferation of the calli on the selection medium.
[0052] 8) New growth that appears on calli on the selection medium
is first transferred to fresh selection medium and allowed to
proliferate. This first selection is best performed when the new
growth is as small as possible in order to ensure that independent
transformants are selected and proliferated in the absence of other
independent transformants.
[0053] 9) After sufficient proliferation,sa small portion of each
of the putatively transformed calli is tested for expression of the
gene of interest, and/or for expression of a detectable marker gene
(e.g., GUS activity). The remaining portions of positive-testing
calli are retained on the selection medium for continued subculture
and proliferation. Subsequently, the transformed calli are
transferred to a regeneration medium containing growth regulators
to promote shoot differentiation.
[0054] 10) When sufficiently, developed, the shoots are transferred
to a second regeneration medium formulated to further stimulate
root growth. After a sufficient growth phase, plantlets are
transferred to either new tissue culture medium or to soil or
equivalent planting media.
[0055] This invention provides transgenic turfgrass produced by the
above-described methods, and also is intended to encompass cells
and tissues of those plants, including, but not limited to, leaves,
stems, shoots, roots, flowers, fruits and seeds. In a preferred
embodiment, seeds of the transgenic plants produced by the methods
of the invention are provided.
[0056] The plants grown from the aforementioned seeds, or seeds
from other turfgrass species or varieties, or the progeny thereof,
all of which are considered within the scope of this invention, are
used in crosses and selection methods to transfer genes of interest
into other turfgrass genotypes, cultivars, varieties and the
like.
[0057] Plants grown from transgenic turfgrass seeds can also be
used to detect the presence of the inserted transgene and vector
sequences using DNA extraction, cleavage by one or more restriction
endonucleases, and analysis, e.g., Southern blotting using probes
derived from the gene or genes of interest. In this manner, the
transfer of foreign genes into progeny of breeding crosses can be
monitored.
[0058] The Agrobacterium-mediated turfgrass transformation system
of the present invention can be used to introduce many genes of
interest into different turfgrass species or varieties.
Accordingly, the present invention provides several specific hybrid
vectors for Agrobacterium-mediated transformation of turfgrass. A
number of gene constructs are of considerable practical utility as
used to create one or more different transgenic turfgrasses. These
include: 1) the gene encoding glucose oxidase from Aspergillus
niger which when expressed in potato provides resistance to
bacterial and fungal pathogens through its production of
H.sub.2O.sub.2 in the plant apoplast (Wu et al., 1995); 2) the gene
encoding citrate synthase from Pseudomonas aeruginosa which when
expressed in the cytoplasm of tobacco provides tolerance to toxic
levels of aluminum in the soil (Manuel et al., 1997); 3) the genes
encoding -9 desaturase from Saccharomyces cerevisiae (Stukey et
al., 1990) and Cryptococcus curvatus (Meesters et al., 1997) and
-11 desaturase from Trichoplosia ni (Knipple et al., 1998). When
the ole1 gene from yeast was expressed in tomato and eggplant, it
provided resistance to fungal pathogens (Wang et al., 1998); 4) the
recently identified gene encoding a plant homolog of the neutrophil
NADPH oxidase which is though to be responsible for the oxidative
burst which is critical in plant defense against pathogens (Keller
et al., 1998); 5) the gene encoding bacteriopsin, a proton pump
from the bacterium, Halobacterium halobium, which has been
demonstrated to protect transgenic tobacco expressing the protein
against plant pathogens (Mittler et al., 1995); and 6) the gene
encoding pokeweed antiviral protein, which is a
ribosome-inactivating protein from the plant Phytolacca americana;
expression of this gene in tobacco provides resistance to viral and
fungal pathogens (Lodge et al., 1993; Zoubenko et al., 1997).
[0059] The following examples are provided to describe the
invention in greater detail. They are intended to illustrate, not
to limit, the invention.
EXAMPLE 1
Construction of Agrobacterium tumefaciens Strain LBA4404,
Containing pSB111SH Superbinary Hybrid Vector
[0060] The vector pTOK233 described by Hiei et al. (1994) for
transformation of rice was not useful for transformation of
creeping bentgrass despite numerous attempts. It was suspected that
the cauliflower mosaic 35S promoter was the source of the problem
in that it appears to be a very weak promoter in turfgrass.
Accordingly, an alternate pair of plasmid vectors was obtained,
namely pSB1 and pSB11 (Japan Tobacco, Inc. Plant Breeding and
Genetics Research Laboratory, 700 Higashibara, Toyoda, Iwata,
Shizuoka, Japan). pSB11 is a 6.3 kb plasmid which contains a
multicloning region with a number of unique restriction sites
between the left and right border sequences delineating the T-DNA
region that will be transferred from Agrobacterium tumefaciens to
the plant genome. Monocot "expression cassettes" expressing
hygromycin B phosphotransferase (to confer hygromycin resistance)
and glucuronidase (GUS, as a scorable reporter) were inserted into
pSB11 into the multiple cloning region between the T-DNA border
sequences. The following strategy was used.
[0061] First, plasmid pAcH1 (provided by Dr. German Spangenberg)
was partially digested with Sac I which yielded several restriction
fragments, of which the 2645 bp fragment was gel-purified. This
fragment contains a 3' modified hph coding sequence (conferring
hygromycin resistance) cloned inframe and downstream from the Act 1
5' promoter sequence, first exon (non-coding), first intron and a
portion of the second, ATG-containing exon of the gene.
Transcription termination signals are provided by the CaMV35S
terminator. This 2645 bp fragment was cloned into the unique Sac I
restriction site between the right and left border sequences of
plasmid pSB11. This plasmid was named pSB11S. Next, a 4175 bp Hind
III fragment was excised from plasmid pAHC25, kindly provided by
Dr. Peter Quail. This fragment contains the promoter, 5'
untranslated exon, and first intron of the maize ubiquitin (Ubi1)
gene fused with the GUS reporter gene coding sequence (derived form
pBI101.2). This Hind III restriction fragment was cloned into the
unique Hind III site of pSB11S, between the right and left border
sequences of the plasmid. The resultant plasmid was named
pSB11SH.
[0062] Next, this intermediate vector (pSB11SH) was electroporated
into Agrobacterium tumefaciens LBA4404 containing the "acceptor
vector" pSB1. Plasmid pSB1 is a "superbinary vector" which carries
a 15.2 kb Kpn I fragment containing additional copies of the vir B,
vir C and vir G virulence genes to enable efficient T-DNA transfer
to monocots. Following homologous recombination between the
"acceptor vector" and the "intermediate vector", a co-integrate
"hybrid vector" was obtained. Bacteria expressing the drug
resistance markers derived from both the acceptor and intermediate
vectors were selected on AB plates containing 10 .mu.g/ml
tetracycline and 50 .mu.g/ml spectinomycin, and the resultant
strains carrying the "hybrid vector" were used in plant
transformations.
EXAMPLE 2
Agrobacterium-Mediated Transformation of Creeping Bentgrass With
Superbinary Hybrid Vector pSB111SH
[0063] In this example, an Agrobacterium transformation system for
creeping bentgrass, Agrostis palustris, is described, which
utilizes the superbinary hybrid test vector described in Example
1
[0064] Media:
MMSG Medium
[0065] Per Liter: TABLE-US-00001 Murashige and Skoog basal salts
4.33 g sucrose 30 g Gamborg vitamins (10000.times.) 1 ml casein
hydrolysate 500 mg dicamba (dichloro-o-anisic acid) 6.6 mg 6 BAP
(6-benzylaminopurine) 0.5 mg Adjust the pH to 5.6 to 5.8. Add
GEL-GRO .TM.* 2.4 g Autoclave and dispense into Petri dishes.
(*GEL-GRO .TM. is gellan gum, an agar substitute, manufactured by
ICN Biomedicals, Inc. If GEL-GRO .TM. is unavailable, agar or an
equivalent agar substitute may be utilized.)
Modified AAM Medium
[0066] Per Liter: TABLE-US-00002 glutamine 875 mg aspartic acid 266
mg arginine 174 mg glycine 7.5 mg sucrose 88.5 mg 2,4-D 1 mg
kinetin 0.2 mg GA.sub.3 0.1 mg Murashige & Skoog vitamins
(1000.times.) 1 ml casamino acids 500 mg glucose 36 g Adjust pH to
5.2 and autoclave. Add acetosyringone to a final concentration of
100 .mu.M.
Regeneration Medium MSO I
[0067] Per Liter: TABLE-US-00003 Murashige & Skoog basal salts
4.33 g Gamborg vitamins (1000.times.) 1 ml sucrose 30 g
myo-inositol 100 mg 6-benzylaminopurine 1 mg Adjust pH to 5.8. Add
GEL-GRO .TM. 2.4 g Autoclave and dispense into plates.
Regeneration Medium MSO II
[0068] Per Liter: TABLE-US-00004 Murashige & Skoog basal salts
4.33 g Gamborg vitamins (1000.times.) 1 ml sucrose 30 g
myo-inositol 100 mg Adjust pH to 5.8. Add GEL-GRO .TM. 2.4 g
Autoclave and dispense into petri dishes and plantcons.
AB Medium
[0069] 1. Prepare a stock of AB Buffer at 20.times. and
autoclave:
[0070] Per Liter: TABLE-US-00005 K.sub.2HPO.sub.4 60 g
NaH.sub.2PO.sub.4 20 g
[0071] 2. Prepare a stock of AB Salts at 20.times. and
autoclave:
[0072] Per Liter: TABLE-US-00006 NH.sub.4Cl 20 g
MgSO.sub.47H.sub.2O 6 g KCl 3 g CaCl.sub.2 0.2 g
FeSO.sub.47H.sub.2O 50 mg
[0073] 3. For 1 liter of medium for plates: [0074] Mix 5 g of
glucose, 15 g of agar, and 900 ml of H.sub.2O and autoclave. [0075]
Add 50 ml of the 20.times. stock of AB Buffer and 50 ml of the
20.times. stock of AB Salts. [0076] Add 10 mg tetracycline and 50
mg spectinomycin. Pour into petri dishes.
[0077] Production of regenerable callus: Mature seeds of creeping
bentgrass (Crenshaw cultivar) were surface-sterilized and plated on
MMSG medium. The plates were kept in the dark at room temperature
for 3-6 weeks. The proliferating calli were selected and
transferred to new MMSG medium on a regular basis. Only callus that
was friable and regenerable was used for transformation. The chosen
callus was transferred to new MMSG medium prior to co-cultivation
to promote active cell division and was used for transformation
within a week after transferring to new plates. The nature of the
callus (i.e., friability, regenerability and active growth) is
believed to play a key role in obtaining efficient
transformation.
[0078] Induction of Agrobacterium tumefaciens with acetosyringone:
Agrobacterium tumefaciens LBA4404, harboring vector pSB111SH, was
streaked from a glycerol stock stored at -80.degree. C. and grown
at 28.degree. C. on plates containing AB medium, supplemented with
10 .mu.g/ml tetracycline and 50 .mu.g/ml spectinomycin. After three
to six days, the cells were scraped from the plate and suspended in
modified AAM medium containing 100 .mu.M acetosyringone to an
OD.sub.660 of approximately 0.5. The bacterial suspension was left
at 25.degree. C. in the dark with shaking for 3.5 hours before
using it for co-cultivation.
[0079] Co-cultivation: A given amount of friable callus was mixed
with the pre-induced Agrobacterium suspension and incubated at room
temperature in the dark for 1.5 hours. Then the contents were
poured into a sterile Buchner-funnel containing a sterile Whatman
filter paper. Mild vacuum was applied to drain the excess
Agrobacterium suspension. Then, the filter was moved to a plate
containing MMSG medium supplemented with 100 .mu.M acetosyringone,
and the plate was stored in the dark at room temperature for three
days.
[0080] Selection and regeneration of transformants: Subsequent to
the three day co-cultivation, the co-cultivated calli were rinsed
with 250 .mu.g/ml cefotaxime to suppress bacterial growth, and the
calli were placed on agar plates containing MMSG medium containing
200 .mu.g/ml hygromycin and 250 .mu.g/ml cefotaxime. The calli were
kept in the dark at room temperature for 6-8 weeks and checked
periodically for proliferation of the calli on hygromycin.
[0081] Subsequently, the hygromycin-resistant calli were placed on
regeneration medium containing hygromycin and cefotaxime. The
proliferating calli were first moved to Regeneration Medium I (MSO
I) containing cefotaxime and hygromycin. These calli were kept in
the dark at room temperature for a week and were then moved to
light for approximately two weeks. The tiny plants were separated
and transferred to deep petri plates containing Regeneration Medium
II (MSO II) to promote root growth. Hygromycin and cefotaxime were
included in the medium to respectively maintain selection pressure
and kill any remaining Agrobacterium cells. After 2-3 weeks, or
when the plants were 1.5-2 cm tall, they were moved to plantcons
containing MSO II without antibiotics. When the plants were
.about.10 cm tall and had developed extensive root systems, they
were transferred to soil and grown in the laboratory for 3-4 weeks
with 12 hours light/day. The plants were transferred to 6'' pots to
the greenhouse, where the temperature was maintained between
21.degree.-25.degree. C. Supplemental lighting added approximately
50 .mu.E m.sup.-2 sec.sup.-1 at canopy level when natural light was
low and provided a minimal light period of 14 hours.
[0082] In connection with the transformation of creeping bentgrass,
it should be noted that the media and protocols described below in
Examples 3 and 4 may also be used to successfully transform and
regenerate creeping bentgrass.
EXAMPLE 3
Agrobacterium-Mediated Transformation of Tall Fescue With
Superbinary Hybrid Vector pSB111SH
[0083] In this example, an Agrobacterium transformation system for
tall fescue, Festuca arundinacea Scheb., is described, which
utilizes the superbinary hybrid test vector described in Example
1.
[0084] Media:
MMSG Medium
[0085] This medium was formulated as described in Example 2.
[0086] Inoculation Medium Per Liter: TABLE-US-00007 Murashige &
Skoog basal salts 440 mg sucrose 30 g dicamba 6.6 mg 6 BAP
(6-benzyl aminopurine) 0.5 mg Gamborg vitamins (1000.times.) 1 ml
casein hydrolysate 500 mg glucose 10 g Adjust pH to 5.7 and
autoclave. Immediately before use add acetosyringone to a final
concentration of 200 .mu.M and pluronic F-68 to a final
concentration of 0.02%.
Co-Cultivation Medium
[0087] Per Liter: TABLE-US-00008 Murashige and Skoog basal salts
4.4 g sucrose 30 g glucose 10 g Gamborg vitamins (1000.times.) 1 ml
casein hydrolysate 500 mg dicamba (dichloro-o-anisic acid) 6.6 mg 6
BAP (6-benzylaminopurine) 0.5 mg Adjust the pH to 5.7 Add GEL-GRO
.TM. 2.4 g Autoclave Add acetosyringone 39.2 mg
Regeneration Medium MSO I
[0088] This medium was formulated and prepared as described in
Example 2.
Regeneration Medium MSO II
[0089] This medium was formulated and prepared as described in
Example 2.
AB Medium
[0090] This medium was formulated and prepared as described in
Example 2.
[0091] Production of regenerable callus: Mature seeds of tall
fescue were surface-sterilized and plated on MMSG medium. The
plates were kept in the dark at room temperature for 3-6 weeks. The
proliferating calli were selected and transferred to new MMSG
medium on a regular basis. Callus chosen for transformation was
transferred to new MMSG medium prior to co-cultivation to promote
active cell division. The nature of the callus (i.e., friability,
regenerability and active growth) is believed to play a key role in
obtaining efficient transformation.
[0092] Preparation of Agrobacterium tumefaciens suspension:
Agrobacterium tumefaciens LBA4404, harboring vector pSB111SH, was
streaked from a glycerol stock stored at -80.degree. C. and grown
at 28.degree. C. on plates containing AB medium, supplemented with
10 .mu.g/ml tetracycline and 50 .mu.g/ml spectinomycin. After three
to six days, the cells were scraped from the plate and suspended in
inoculation medium containing 200 .mu.M acetosyringone and 0.02%
pluronic Fy68 to an OD.sub.660 between 0.5 and 0.8.
[0093] Co-cultivation of callus with Agrobacterium: The friable
callus chosen for transformation was placed in a sterile tube and
mixed with 30 ml of Agrobacterium suspension. The tube was capped
and covered with aluminum foil, and the contents were mixed by
inversion and gently shaken for about 1.5 hr. The contents of the
tube were poured into a Buchner funnel, fitted with Whatman filter.
Mild vacuum was applied to flask. The filter disks containing
callus with Agrobacterium were moved to co-cultivation plates
containing acetosyringone (200 .mu.M) and glucose at 10 g/l. The
plates were sealed with parafilm and placed in the dark at room
temperature for three days.
[0094] Selection and regeneration of transformants:. Subsequent to
the three day co-cultivation, the co-cultivated calli were: rinsed
with 250 .mu.g/ml cefotaxime solution to suppress bacterial growth,
and the calli were placed on MMSG medium containing 200 .mu.g/ml
hygromycin and 250 .mu.g/ml cefotaxime. The calli were kept in the
dark at room temperature for 6-8 weeks and checked periodically for
proliferation of the calli on hygromycin. The hygromycin-resistant
calli were moved to new MMSG plates with hygromycin and kept in the
dark at room temperature until well proliferated; Then a portion of
the hygromycin-resistant callus was tested for GUS activity to
ensure that transformation had occurred.
[0095] Portions of the putatively transformed calli were then moved
to Regeneration Medium I (MSO I) containing cefotaxime and
hygromycin and kept in the dark room at room temperature for a week
and were subsequently moved to the light for regeneration. The tiny
shoots were separated and transferred to deep petri plates
containing Regeneration Medium II (MSO II) to promote root growth
and hygromycin and cefotaxime to maintain respectively selection of
the transformants and kill any remaining Agrobacterium.
EXAMPLE 4
Agrobacterium-Mediated Transformation of Velvet Bentgrass With
Superbinary Hybrid Vector pSB111SH
[0096] In this example, an Agrobacterium transformation system for
velvet bentgrass, Agrostis canina L., is described, which utilizes
the superbinary hybrid test vector described in Example 1.
[0097] Media: All media were formulated and prepared as described
in Example 3.
[0098] Production of regenerable callus: Mature seeds of velvet
bentgrass were surface-sterilized and plated on MMSG medium. The
plates were kept in the dark at room temperature for 3-6 weeks. The
proliferating calli were selected and transferred to new MMSG
medium on a regular basis. Callus chosen for transformation was
transferred to new MMSG medium prior to co-cultivation to promote
active cell division The nature of the callus (i.e., friability,
regenerability and active growth) is believed to play a key role in
obtaining efficient transformation.
[0099] Preparation of Agrobacterium tumefaciens Suspension:
Agrobacterium tumefaciens LBA4404, harboring vector pSB111SH, was
streaked from a glycerol stock stored at -80.degree. C. and grown
at 28.degree. C. on plates containing AB medium, supplemented with
10 .mu.g/ml tetracycline and 50 .mu.g/ml spectinomycin. After three
to six days, the bacterial lawn was scraped from one (82 mm
diameter) plate and suspended in 6 ml inoculation medium containing
200 .mu.M acetosyringone. The bacterial suspension was left at
28.degree. C. in the dark with shaking overnight. In the morning
acetosyringone was added to a final concentration of 400 .mu.M and
pluronic F-68 to 0.02%.
[0100] Co-cultivation: A sterile filter was placed on a
co-cultivation plate containing acetosyringone (200 .mu.M) and
glucose (10 gm/l). Onto this filter was placed one large clump of
friable callus which was then gently broken up and dispensed evenly
over the filter. About 500 .mu.l of Agrobacterium suspension was
pipetted evenly onto the callus. The plates were sealed with
parafilm and placed in the dark at room temperature for three
days.
[0101] Selection and regeneration of transformants: Subsequent to
the three day co-cultivation, the co-cultivated calli were rinsed
with 250 .mu.g/ml cefotaxime solution to suppress bacterial growth,
and the calli were placed on MMSG medium containing 200 .mu.g/ml
hygromycin and 250 .mu.g/ml cefotaxime. The calli were kept in the
dark at room temperature for 6-8 weeks and checked periodically for
proliferation of the calli on hygromycin. The hygromycin-resistant
calli were moved to new MMSG plates with hygromycin and kept in the
dark at room temperature until well proliferated. Then a portion of
the hygromycin-resistant callus was tested for GUS activity to
ensure that transformation had occurred.
[0102] Portions of the putatively transformed calli were then moved
to Regeneration Medium I (MSO I) containing cefotaxime and
hygromycin and were kept in the dark room at room temperature.
After one week, they were moved to the light for regeneration. The
tiny shoots were separated and transferred to deep petri plates
containing Regeneration Medium II (MSO II) to promote root growth
and hygromycin and cefotaxime to maintain respectively selection
pressure and kill any remaining Agrobacterium cells.
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[0126] While certain of the preferred embodiments of the present
invention have been described and specifically exemplified above,
it is not intended that the invention be limited to such
embodiments. Various modifications may be made thereto without
departing from the scope and spirit of the present invention, as
set forth in the following claims.
* * * * *