U.S. patent application number 10/417650 was filed with the patent office on 2003-10-30 for novel agrobacterium-mediated plant transformation method.
Invention is credited to Armstrong, Charles L., Rout, Jyoti R..
Application Number | 20030204875 10/417650 |
Document ID | / |
Family ID | 23433702 |
Filed Date | 2003-10-30 |
United States Patent
Application |
20030204875 |
Kind Code |
A1 |
Armstrong, Charles L. ; et
al. |
October 30, 2003 |
Novel agrobacterium-mediated plant transformation method
Abstract
The present invention relates to a novel transformation system
for generating transformed plants with lower copy inserts and
improved transformation efficiency. In particular, the invention
relates to the use of Agrobacterium growth inhibiting agents during
the Agrobacterium-mediated transformation process that suppress
Agrobacterium growth and reduce T-DNA transfer to the target plant
genome.
Inventors: |
Armstrong, Charles L.; (St.
Charles, MO) ; Rout, Jyoti R.; (Middleton,
WI) |
Correspondence
Address: |
MONSANTO COMPANY
800 N. LINDBERGH BLVD.
ATTENTION: G.P. WUELLNER, IP PARALEGAL, (E2NA)
ST. LOUIS
MO
63167
US
|
Family ID: |
23433702 |
Appl. No.: |
10/417650 |
Filed: |
April 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10417650 |
Apr 17, 2003 |
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09364254 |
Jul 29, 1999 |
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6603061 |
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Current U.S.
Class: |
800/294 |
Current CPC
Class: |
C12N 15/8205 20130101;
A01H 4/001 20130101 |
Class at
Publication: |
800/294 |
International
Class: |
A01H 001/00; C12N
015/82 |
Claims
What is claimed is:
1. A method of transforming a plant cell or plant tissue using an
Agrobacterium mediated process comprising the steps of: inoculating
a transformable plant cell or tissue with Agrobacterium containing
at least one genetic component capable of being transferred to the
plant cell or tissue in the presence of at least one growth
inhibiting agent; co-culturing the transformable plant cell or
tissue after inoculation in a media capable of supporting growth of
plant cells or tissue expressing the genetic component, said media
not containing a growth inhibiting agent; selecting transformed
plant cells or tissue; and regenerating a transformed plant
expressing the genetic component from the selected transformed
plant cells or tissue.
2. The method of claim 1 wherein the presence of the growth
inhibiting agent during inoculation reduces the T-DNA transfer
process of the Agrobacterium.
3. The method of claim 1 wherein the growth inhibiting agent is
selected from the group consisting of antibiotics, compounds
containing a heavy metal ion, and proteins, nucleic acids, cell
extracts, growth regulators, or secondary metabolites capable of
inhibiting or suppressing the growth of Agrobacterium.
4. The method of claim 3 wherein the compound containing a heavy
metal ion contains silver, potassium, manganese or cadmium.
5. The method of claim 4 wherein the heavy metal ion is silver.
6. The method of claim 3 wherein the growth inhibiting agent is
silver nitrate.
7. The method of claim 6 wherein the concentration of silver
nitrate is from about 5 .mu.M to about 50 .mu.M.
8. The method of claim 7 wherein the concentration of silver
nitrate is about 20 .mu.M.
9. The method of claim 3 wherein the growth inhibiting agent is
silver thiosulfate.
10. The method of claim 9 wherein the concentration of silver
thiosulfate is from about 5 .mu.M to about 50 .mu.M.
11. The method of claim 10 wherein the concentration of silver
thiosulfate is 20 .mu.M.
12. The method of claim 3 wherein the growth inhibiting agent is an
antibiotic.
13. The method of claim 12 wherein the antibiotic is
carbenicillin.
14. The method of claim 3 wherein the growth inhibiting agent is a
nucleic acid capable of suppressing Agrobacterium cell growth and
the T-DNA transfer process.
15. The method of claim 1 wherein the transformable plant cell or
tissue is from a monocotyledonous plant.
16. The method of claim 1 wherein the transformable plant cell or
tissue is from a dicotyledonous plant.
17. The method of claim 15 wherein the monocotyledonous plant is a
cereal.
18. The method of claim 16 wherein the monocotyledonous plant is
corn, wheat, or rice.
19. The method of claim 17 wherein the dicotyledonous plant is
soybean, cotton, canola, or sunflower.
20. The method of claim 18 wherein the monocotyledonous plant is
corn.
21. The method of claim 18 wherein the monocotyledonous plant is
wheat.
22. The method of claim 18 wherein the monocotyledonous plant is
rice.
23. A method of transforming a plant cell or plant tissue using an
Agrobacterium mediated process comprising the steps of: inoculating
a transformable plant cell or tissue with Agrobacterium containing
at least one genetic component capable of being transferred to the
plant cell or tissue; co-culturing the transformable plant cell or
tissue after inoculation in a media capable of supporting growth of
plant cells or tissue expressing the genetic component, said media
further containing a growth inhibiting agent; selecting transformed
plant cells or tissue; and regenerating a transformed plant
expressing the genetic component from the selected transformed
cells or tissue.
24. The method of claim 23 wherein the presence of the growth
inhibiting agent during co-culture reduces the T-DNA transfer
process of the Agrobacterium.
25. The method of claim 23 wherein the growth inhibiting agent is
selected from the group consisting of antibiotics, compounds
containing a heavy metal ion, and proteins, nucleic acids, cell
extracts, growth regulators, or secondary metabolites capable of
inhibiting or suppressing the growth of Agrobacterium.
26. The method of claim 25 wherein the compound containing a heavy
metal ion contains silver, potassium, manganese or cadmium.
27. The method of claim 26 wherein the heavy metal ion is
silver.
28. The method of claim 25 wherein the growth inhibiting agent is
silver nitrate.
29. The method of claim 28 wherein the concentration of silver
nitrate is from about 5 .mu.M to about 50 .mu.M.
30. The method of claim 29 wherein the concentration of silver
nitrate is about 20 .mu.M.
31. The method of claim 25 wherein the growth inhibiting agent is
silver thiosulfate.
32. The method of claim 31 wherein the concentration of silver
thiosulfate is from about 5 .mu.M to about 50 .mu.M.
33. The method of claim 32 wherein the concentration of silver
thiosulfate is 20 .mu.M.
34. The method of claim 25 wherein the growth inhibiting agent is
an antibiotic.
35. The method of claim 34 wherein the antibiotic is
carbenicillin.
36. The method of claim 25 wherein the growth inhibiting agent is a
nucleic acid capable of suppressing Agrobacterium cell growth and
the T-DNA transfer process.
37. The method of claim 23 wherein the transformable plant cell or
tissue is from a monocotyledonous plant.
38. The method of claim 23 wherein the transformable plant cell or
tissue is from a dicotyledonous plant.
39. The method of claim 37 wherein the monocotyledonous plant is a
cereal.
40. The method of claim 39 wherein the monocotyledonous plant is
corn, wheat, or rice.
41. The method of claim 38 wherein the dicotyledonous plant is
soybean, cotton, canola, or sunflower.
42. The method of claim 40 wherein the monocotyledonous plant is
corn.
43. The method of claim 40 wherein the monocotyledonous plant is
wheat.
44. The method of claim 40 wherein the monocotyledonous plant is
rice.
45. A method of transforming a plant cell or plant tissue using an
Agrobacterium mediated process comprising the steps of: inoculating
a transformable plant cell or tissue with Agrobacterium containing
at least one genetic component capable of being transferred to the
plant cell or tissue in the presence of at least one growth
inhibiting agent; co-culturing the transformable plant cell or
tissue after inoculation in a media capable of supporting growth of
the plant cells or tissue expressing the genetic component, said
media further containing a growth inhibiting agent; selecting
transformed plant cells or tissue; and regenerating a transformed
plant expressing the genetic component from the selected
transformed cells or tissue.
46. The method of claim 45 wherein the presence of the growth
inhibiting agent during inoculation and co-culture reduces the
T-DNA transfer process of the Agrobacterium.
47. The method of claim 45 wherein the growth inhibiting agent is
selected from the group consisting of antibiotics, compounds
containing a heavy metal ion, and proteins, nucleic acids, cell
extracts, growth regulators, or secondary metabolites capable of
inhibiting or suppressing the growth of Agrobacterium.
48. The method of claim 47 wherein the compound containing a heavy
metal ion contains silver, potassium, manganese or cadmium.
49. The method of claim 48 wherein the heavy metal ion is
silver.
50. The method of claim 47 wherein the growth inhibiting agent is
silver nitrate.
51. The method of claim 50 wherein the concentration of silver
nitrate is from about 5 .mu.M to about 50 .mu.M.
52. The method of claim 51 wherein the concentration of silver
nitrate is about 20 .mu.M.
53. The method of claim 47 wherein the growth inhibiting agent is
silver thiosulfate.
54. The method of claim 53 wherein the concentration of silver
thiosulfate is from about 5 .mu.M to about 50 .mu.M.
55. The method of claim 54 wherein the concentration of silver
thiosulfate is 20 .mu.M.
56. The method of claim 47 wherein the growth inhibiting agent is
an antibiotic.
57. The method of claim 56 wherein the antibiotic is
carbenicillin.
58. The method of claim 47 wherein the growth inhibiting agent is a
nucleic acid capable of suppressing Agrobacterium cell growth and
the T-DNA transfer process.
59. The method of claim 45 wherein the transformable plant cell or
tissue is from a monocotyledonous plant.
60. The method of claim 45 wherein the transformable plant cell or
tissue is from a dicotyledonous plant.
61. The method of claim 59 wherein the monocotyledonous plant is a
cereal.
62. The method of claim 61 wherein the monocotyledonous plant is
corn, wheat, or rice.
63. The method of claim 60 wherein the dicotyledonous plant is
soybean, cotton, canola, or sunflower.
64. The method of claim 62 wherein the monocotyledonous plant is
corn.
65. The method of claim 62 wherein the monocotyledonous plant is
wheat.
66. The method of claim 62 wherein the monocotyledonous plant is
rice.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to the field of plant
biotechnology. More specifically, it concerns methods of
incorporating genetic components into a plant comprising a T-DNA
transfer process. In particular, provided herein are systems for
genetically transforming monocotyledonous plants including corn,
rice, and wheat.
[0002] The method comprises novel conditions during the
inoculation, co-culture, or infiltration of Agrobacterium with a
transformable plant cell or tissue. Exemplary methods include an
improved method using a bacterial growth suppressing agent during
the Agrobacterium-mediated transformation process. The improved
method can be used for introducing nucleic acids into transformable
cells or tissues using a variety of selectable and/or screenable
marker systems, and with a number of different plant species. The
present invention also provides transgenic plants, in particular,
corn, rice, and wheat. In other aspects, the invention relates
to-the production of stably transformed plants, gametes, and
offspring from these plants.
[0003] During the past decade, it has become possible to transfer
genes from a wide range of organisms to crop plants by recombinant
DNA technology. This advance has provided enormous opportunities to
improve plant resistance to pests, disease and herbicides, and to
modify biosynthetic processes to change the quality of plant
products (Knutson et al., 1992; Piorer et al., 1992). However, the
availability of efficient Agrobacterium-mediated transformation
methods suitable for high capacity production of economically
important plants is limited. In particular, a novel culture system
that generates reproducible transformants with a simple integration
pattern of the introduced DNA into the host genome, more
specifically, the integration of a low copy number (one to two
copies) of the introduced DNA is needed.
[0004] There have been many methods attempted for plant
transformation, but only a few methods are highly efficient.
Moreover, few methods are both highly efficient and result in
transformants with simple integration pattern and low copy number
of the introduced DNA. Copy number refers to the number of complete
or incomplete copies of T-DNA introduced in host cell. The
technologies for the introduction of DNA into cells are well known
to those of skill in the art and can be divided into categories
including but not limited to: (1) chemical methods (Graham and van
der Eb, 1973); (2) physical methods such as microinjection
(Capecchi, 1980), electroporation (Fromm et al., 1985; U.S. Pat.
No. 5,384,253) and the gene gun (Christou, 1992; Fynan et al.,
1993); (3) viral vectors (Clapp, 1993; Lu et al., 1993; Eglitis and
Anderson, 1988);(4) receptor-mediated mechanisms (Curiel et al.,
1992); and (5) Agrobacterium-mediated plant transformation
methods.
[0005] Until recently, the methods employed for some monocot
species included direct DNA transfer into isolated protoplasts and
microprojectile-mediated DNA delivery (Fromm et al, 1990). The
protoplast methods have been widely used in rice, where DNA is
delivered to the protoplasts through liposomes, PEG, and
electroporation. While a large number of transgenic plants have
been recovered in several laboratories (Datta et al., 1990), the
protoplast methods require the establishment of long-term
embryogenic suspension cultures. Some regenerants from protoplasts
are infertile and phenotypically abnormal due to the long-term
suspension culture (Davey et al., 1991; Rhodes et al., 1988). U.S.
Pat. No. 5,631,152 describes a rapid and efficient microprojectile
bombardment method for the transformation and regeneration of
monocots and dicots.
[0006] To date, microparticle- and Agrobacterium-mediated gene
delivery are the two most commonly used plant transformation
methods. Microparticle-mediated transformation refers to the
delivery of DNA coated onto microparticles that are propelled into
target tissues by several methods. This method can result in
transgenic events with a higher copy number, complex integration
patterns, and fragmented inserts. Agrobacterium-mediated plant
transformation can also result in transformed plants with multiple
copies of inserts and complex integration patterns. A reduction in
copy number can result from a decrease in the frequency of T-DNA
transfer. Accordingly, novel culture conditions can be manipulated
to impact the frequency of T-DNA transfer and can produce
transformation events containing the optimum number of copies of
the introduced DNA.
[0007] A reproducible Agrobacterium-mediated method that
consistently results in low copy number inserts and is applicable
to a broad number of plant species is desirable for a number of
reasons. For example, the presence of multiple inserts can lead to
a phenomenon known as gene silencing which can occur by several
mechanisms including but not limited to recombination between the
multiple copies which can lead to subsequent gene loss. Also,
multiple copies can cause reduced levels of expression of the gene
which in turn can result in the reduction of the characteristic(s)
conferred by the gene product(s). Despite the number of
transformation methods available for specific plant systems, it
would be advantageous to have a method of introducing genes into
plants that is applicable to various crops and a variety of
transformable tissues.
[0008] Agrobacterium-mediated transformation is achieved through
the use of a genetically engineered soil bacterium belonging to the
genus Agrobacterium. Several Agrobacterium species mediate the
transfer of a specific DNA known as "T-DNA", that can be
genetically engineered to carry any desired piece of DNA into many
plant species. The major events marking the process of T-DNA
mediated pathogenesis are: induction of virulence genes, processing
and transfer of T-DNA. This process is the subject of many reviews
(Ream, 1989; Howard and Citovsky, 1990; Kado, 1991; Hooykaas and
Schilperoort, 1992; Winnans, 1992; Zambryski, 1992; Gelvin, 1993;
Binns and Howitz, 1994; Hooykaas and Beijersbergen 1994; Lessl and
Lanka, 1994; Zupan and Zambryski, 1995).
[0009] Agrobacterium-mediated genetic transformation of plants
involves several steps. The first step, in which the Agrobacterium
and plant cells are first brought into contact with each other, is
generally called "inoculation". Following the inoculation step, the
Agrobacterium and plant cells/tissues are usually grown together
for a period of several hours to several days or more under
conditions suitable for growth and T-DNA transfer. This step is
termed "co-culture". Following co-culture and T-DNA delivery, the
plant cells are often treated with bacteriocidal and-or
bacteriostatic agents to kill the Agrobacterium. If this is done in
the absence of any selective agents to promote preferential growth
of transgenic versus non-transgenic plant cells, then this is
typically referred to as the "delay" step. If done in the presence
of selective pressure favoring tranasgenic plant cells, then it is
referred to as a "selection" step. When a "delay" is used, it is
followed by one or more "selection" steps. Both the "delay" and
"selection" steps typically include bacteriocidal and-or
bacteriostatic agents to kill any remaining Agrobacterium cells
because the growth of Agrobacterium cells is undesirable after the
infection (inoculation and co-culture) process.
[0010] Although transgenic plants produced through
Agrobacterium-mediated transformation generally contain a simple
integration pattern as compared to microparticle-mediated genetic
transformation, a wide variation in copy number and insertion
patterns exists (Jones et al, 1987; Jorgensen et al., 1987; Deroles
and Gardner, 1988). Moreover, even within a single plant genotype,
different patterns of T-DNA integration are possible based on the
type of explant and transformation system used (Grevelding et al.,
1993). Factors that regulate T-DNA copy number are poorly
understood. A reproducible, broadly applicable method to increase
the efficiency of producing plants with a low copy number, and
preferably a single copy of the T-DNA would be highly desirable to
those practicing in the art.
[0011] Recently, monocot species have been successfully transformed
via Agrobacterium-mediated transformation. WO 97/48814 discloses
processes for producing stably transformed fertile wheat. The
method describes the recovery of transgenic, wheat plants within a
short period of time using a variety of explants.
Agrobacterium-mediated transformation provides a viable alternative
to bombardment methods and the method also allows more efficient
molecular characterization of transgenic lines. The present
invention is an improved Agrobacterium-mediated transformation
method that relies on the control of Agrobacterium growth during
the transformation process. More specifically, the present
invention focuses on controlling Agrobacterium growth in the stages
of Agrobacterium-mediated transformation during which T-DNA
transfer can occur.
[0012] The major deficiencies in current plant transformation
systems utilizing Agrobacterium-mediated methods include but are
not limited to the production efficiency of the system, and
transformation difficulties due to genotype or species diversity
and explant limitations. WO 94/00977 describes a method for
transforming monocots that depends on the use of freshly cultured
immature embryos for one monocot and cultured immature embryos or
callus for a different monocot. In either system, the explants must
be freshly isolated, and the method is labor intensive, genotype-,
and explant-limited. Other reports rely on the use of specific
strains or vectors to achieve high efficiency transformation. In
one report, a specific super-binary vector must be used in order to
achieve high-efficiency transformation (Ishida et al., 1996).
[0013] Despite the number of transformation methods in the art, few
methods have been developed that are broadly applicable to
genotypes of a single crop species as well as to genotypes of other
crop species. What is lacking in the art is an
Agrobacterium-mediated plant transformation system that is
efficient, reproducible, applicable to a number of plant systems,
and a transformation system that effectively results in transformed
plants with a simple integration pattern and a low copy number. The
present invention provides novel culture conditions using one or
more bacterial growth inhibiting agents during inoculation and
co-culture of Agrobacterium with a transformable plant cell or
tissue that result in increased transformation efficiencies and a
low copy number of the introduced genetic component in several
plant systems. The method of the present invention consistently
results in desired transgenic events with a low number of inserts
and reduces the need to screen hundreds of lines for identification
of the optimal commercial line for breeding and introduction of
improved germplasm to plant breeders, growers, and consumers. The
present invention thus provides a novel improvement compared to
existing Agrobacterium-mediated transformation methods.
SUMMARY OF THE INVENTION
[0014] The present invention provides novel methods for the stable
and efficient transformation of plants under conditions that
inhibit the growth of Agrobacterium cells during the transformation
process.
[0015] In one aspect the present invention provides a novel method
of transforming a plant cell or plant tissue with Agrobacterium by
inoculating a transformable cell or tissue containing at least one
genetic component capable of being transferred to the plant cell or
tissue in the presence of at least one growth inhibiting agent,
co-culturing in the presence or absence of the growth inhibiting
agent, selecting a transformed plant cell or tissue, and
regenerating a transformed plant expressing the genetic component
from the selected plant cells or tissues.
[0016] In one embodiment, the growth inhibiting agent comprises a
compound containing a heavy metal such as silver, or an antibiotic
such as carbenicillin, or a nucleic acid, or protein capable of
inhibiting or suppressing the growth of Agrobacterium cells and the
growth inhibiting agent is present during the inoculation step in
the transformation process and not in the co-culture step.
[0017] In another embodiment, the growth inhibiting agent that is
inhibitory to Agrobacterium cell growth is present during the
inoculation and co-culture steps in the transformation process.
[0018] In another embodiment, the growth inhibiting agent that is
inhibitory to Agrobacterium cell growth is absent during the
inoculation step, but present in the co-culture step in the
transformation process.
[0019] In still another embodiment the invention relates to the
presence of at least one Agrobacterium growth inhibiting agent
during the inoculation process in an amount sufficient to suppress
Agrobacterium growth and reduce T-DNA transfer, thus favoring low
copy insertions of the introduced DNA.
[0020] Still another aspect of the present invention relates to
transformed plants produced by inoculating a transformable cell or
tissue containing at least one genetic component capable of being
transferred to the plant cell or tissue in the presence of at least
one growth inhibiting agent, co-culturing in the presence or
absence of the growth inhibiting agent, selecting a transformed
plant cell or tissue and regenerating a transformed plant
expressing the genetic component from the selected plant cells or
tissues.
[0021] Yet another aspect of the present invention relates to any
seeds, or progeny of the transformed plants produced by the method
of the present invention.
[0022] Further objects, advantages and aspects of the present
invention will become apparent from the accompanying figures and
description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is a plasmid map of pMON30100
[0024] FIG. 2 is a plasmid map of pMON18365
[0025] FIG. 3 is a plasmid map of pMON25457
[0026] FIG. 4 is a plasmid map of pMON25492
[0027] FIG. 5 is a plasmid map of pMON32092
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention can be used with any plant species. It
is particularly useful for monocot species. Particularly preferred
species for practice of the present invention include corn, wheat,
and rice.
[0029] The present invention provides a transgenic plant and a
method for transformation of plant cells or tissues and recovery of
the transformed cells or tissues into a differentiated transformed
plant. To initiate a transformation process in accordance with the
present invention, it is first necessary to select genetic
components to be inserted into the plant cells or tissues. Genetic
components can include any nucleic acid that is introduced into a
plant cell or tissue using the method according to the invention.
Genetic components can include non-plant DNA, plant DNA or
synthetic DNA.
[0030] In a preferred embodiment, the genetic components are
incorporated into a DNA composition such as a recombinant,
double-stranded plasmid or vector molecule comprising at least one
or more of following types of genetic components:
[0031] (a) a promoter that functions in plant cells to cause the
production of an RNA sequence,
[0032] (b) a structural DNA sequence that causes the production of
an RNA sequence that encodes a product of agronomic utility,
and
[0033] (c) a 3' non-translated DNA sequence that functions in plant
cells to cause the addition of polyadenylated nucleotides to the 3'
end of the RNA sequence.
[0034] The vector may contain a number of genetic components to
facilitate transformation of the plant cell or tissue and regulate
expression of the desired gene(s). In one preferred embodiment, the
genetic components are oriented so as to express a mRNA, that in
one embodiment can be translated into a protein. The expression of
a plant structural coding sequence (a gene, cDNA, synthetic DNA, or
other DNA) that exists in double-stranded form involves
transcription of messenger RNA (mRNA) from one strand of the DNA by
RNA polymerase enzyme and subsequent processing of the mRNA primary
transcript inside the nucleus. This processing involves a 3'
non-translated region that adds polyadenylated nucleotides to the
3' ends of the mRNA.
[0035] Means for preparing plasmids or vectors containing the
desired genetic components are well known in the art. Vectors used
to transform plants and methods of making those vectors are
described in U.S. Pat. Nos. 4,971,908, 4,940,835, 4,769,061 and
4,757,011, the entirety of which are incorporated herein by
reference. Vectors typically consist of a number of genetic
components, including but not limited to regulatory elements such
as promoters, leaders, introns, and terminator sequences.
Regulatory elements are also referred to as cis- or
trans-regulatory elements, depending on the proximity of the
element to the sequences or gene(s) they control.
[0036] Transcription of DNA into mRNA is regulated by a region of
DNA usually referred to as the "promoter". The promoter region
contains a sequence of bases that signals RNA polymerase to
associate with the DNA, and to initiate the transcription into mRNA
using one of the DNA strands as a template to make a corresponding
complementary strand of RNA.
[0037] A number of promoters that are active in plant cells have
been described in the literature. Such promoters would include but
are not limited to the nopaline synthase (NOS) and octopine
synthase (OCS) promoters that are carried on tumor-inducing
plasmids of Agrobacterium tumefaciens, the caulimovirus promoters
such as the cauliflower mosaic virus (CaMV) 19S and 35S promoters
and the figwort mosaic virus (FMV) 35S promoter, the enhanced
CaMV35S promoter (e35S), the light-inducible promoter from the
small subunit of ribulose bisphosphate carboxylase (ssRUBISCO, a
very abundant plant polypeptide). All of these promoters have been
used to create various types of DNA constructs that have been
expressed in plants. See, for example PCT publication WO 84/02913
(Rogers et al., Monsanto, herein incorporated by reference in its
entirety).
[0038] Promoter hybrids can also be constructed to enhance
transcriptional activity (Hoffman, U.S. Pat. No. 5,106,739), or to
combine desired transcriptional activity, inducibility and tissue
specificity or developmental specificity. Promoters that function
in plants include but are not limited to promoters that are
inducible, viral, synthetic, constitutive as described (Poszkowski
et al., 1989; Odell et al., 1985), and temporally regulated,
spatially regulated, and spatio-temporally regulated (Chau et al.,
1989). Other promoters that are tissue-enhanced, tissue-specific,
or developmentally regulated are also known in the art and
envisioned to have utility in the practice of this invention.
[0039] Promoters may be obtained from a variety of sources such as
plants and plant DNA viruses and include, but are not limited to
the CaMV35S and FMV35S promoters and promoters isolated from plant
genes such as ssRUBISCO genes. As described below, it is preferred
that the particular promoter selected should be capable of causing
sufficient expression to result in the production of an effective
amount of the gene product of interest.
[0040] The promoters used in the DNA constructs (i.e.
chimeric/recombinant plant genes) of the present invention may be
modified, if desired, to affect their control characteristics.
Promoters can be derived by means of ligation with operator
regions, random or controlled mutagenesis, etc. Furthermore, the
promoters may be altered to contain multiple "enhancer sequences"
to assist in elevating gene expression. Examples of such enhancer
sequences have been reported by Kay et al. (1987).
[0041] The mRNA produced by a DNA construct of the present
invention may also contains a 5' non-translated leader sequence.
This sequence can be derived from the promoter selected to express
the gene, and can be specifically modified so as to increase
translation of the mRNA. The 5' non-translated regions can also be
obtained from viral RNAs, from suitable eukaryotic genes, or from a
synthetic gene sequence (Griffiths, et al., 1993) Such "enhancer"
sequences may be desirable to increase or alter the translational
efficiency of the resultant mRNA. The present invention is not
limited to constructs wherein the non-translated region is derived
from both the 5' non-translated sequence that accompanies the
promoter sequence. Rather, the non-translated leader sequence can
be derived from unrelated promoters or genes. (see, for example
U.S. Pat. No. 5,362,865). Other genetic components that serve to
enhance expression or affect transcription or translational of a
gene are also envisioned as genetic components.
[0042] The 3' non-translated region of the chimeric constructs
should contain a transcriptional terminator, or an element having
equivalent function, and a polyadenylation signal that functions in
plants to cause the addition of polyadenylated nucleotides to the
3' end of the RNA. Examples of suitable 3' regions are (1) the 3'
transcribed, non-translated regions containing the polyadenylation
signal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as
the nopaline synthase (NOS) gene, and (2) plant genes such as the
soybean storage protein genes and the small subunit of the
ribulose-1,5-bisphosphate carboxylase (ssRUBISCO) gene. An example
of a preferred 3' region is that from the ssRUBISCO E9 gene from
pea (Fischhoff et al., European Patent Application 0385 962, herein
incorporated by reference in its entirety).
[0043] Typically, DNA sequences located a few hundred base pairs
downstream of the polyadenylation site serve to terminate
transcription. The DNA sequences are referred to herein as
transcription-termination regions. The regions are required for
efficient polyadenylation of transcribed messenger RNA (mRNA) and
are known as 3' non-translated regions. RNA polymerase transcribes
a coding DNA sequence through a site where polyadenylation
occurs.
[0044] In one preferred embodiment, the vector contains a
selectable, screenable, or scoreable marker gene. These genetic
components are also referred to herein as functional genetic
components, as they produce a product that serves a function in the
identification of a transformed plant, or a product of agronomic
utility. The DNA that serves as a selection device functions in a
regenerable plant tissue to produce a compound that would confer
upon the plant tissue resistance to an otherwise toxic compound.
Genes of interest for use as a selectable, screenable, or scorable
marker would include but are not limited to GUS, green fluorescent
protein (GFP), luciferase (LUX), antibiotic or herbicide tolerance
genes. Examples of transposons and associated antibiotic resistance
genes include the transposons Tns (bla), Tn5 (nptII), Tn7 (dhfr),
penicillins, kanamycin (and neomycin, G418, bleomycin);
methotrexate (and trimethoprim); chloramphenicol; kanamycin and
tetracycline.
[0045] Characteristics useful for selectable markers in plants have
been outlined in a report on the use of microorganisms (Advisory
Committee on Novel Foods and Processes, July 1994). These
include:
[0046] i) stringent selection with minimum number of nontransformed
tissues;
[0047] ii) large numbers of independent transformation events with
no significant interference with the regeneration;
[0048] iii) application to a large number of species; and
[0049] iv) availability of an assay to score the tissues for
presence of the marker.
[0050] As mentioned, several antibiotic resistance markers satisfy
these criteria, including those resistant to kanamycin (nptII),
hygromycin B (aph IV) and gentamycin (aac3 and aacC4).
[0051] A number of selectable marker genes are known in the art and
can be used in the present invention (see for example Wilmink and
Dons, 1993). Particularly preferred selectable marker genes for use
in the present invention would include genes that confer resistance
to compounds such as antibiotics like kanamycin (Dekeyser et al.,
1989), and herbicides like glyphosate (Della-Cioppa et al., 1987).
Other selection devices can also be implemented including but not
limited to tolerance to phosphinothricin, bialaphos, and positive
selection mechanisms (Joersbo et al., 1998) and would still fall
within the scope of the present invention.
[0052] The present invention can be used with any suitable plant
transformation plasmid or vector containing a selectable or
screenable marker and associated regulatory elements as described,
along with one or more nucleic acids expressed in a manner
sufficient to confer a particular desirable trait. Examples of
suitable structural genes of agronomic interest envisioned by the
present invention would include but are not limited to genes for
insect or pest tolerance, herbicide tolerance, genes for quality
improvements such as yield, nutritional enhancements, environmental
or stress tolerances, or any desirable changes in plant physiology,
growth, development, morphology or plant product(s).
[0053] Alternatively, the DNA coding sequences can effect these
phenotypes by encoding a non-translatable RNA molecule that causes
the targeted inhibition of expression of an endogenous gene, for
example via antisense- or cosuppression-mediated mechanisms (see,
for example, Bird et al., 1991). The RNA could also be a catalytic
RNA molecule (i.e., a ribozyme) engineered to cleave a desired
endogenous mRNA product (see for example, Gibson and Shillitoe,
1997). Thus, any gene that produces a protein or mRNA that
expresses a phenotype or morphology change of interest are useful
for the practice of the present invention.
[0054] Exemplary nucleic acids that may be introduced by the
methods encompassed by the present invention include for example,
DNA sequences or genes from another species, or even genes or
sequences that originate with or are present in the same species,
but are incorporated into recipient cells by genetic engineering
methods rather than classical reproduction or breeding techniques.
However, the term exogenous is also intended to refer to genes that
are not normally present in the cell being transformed, or perhaps
simply not present in the form, structure, etc., as found in the
transforming DNA segment or gene, or genes that are normally
present yet that one desires, e.g., to have over-expressed. Thus,
the term "exogenous" gene or DNA is intended to refer to any gene
or DNA segment that is introduced into a recipient cell, regardless
of whether a similar gene may already be present in such a cell.
The type of DNA included in the exogenous DNA can include DNA that
is already present in the plant cell, DNA from another plant, DNA
from a different organism, or a DNA generated externally, such as a
DNA sequence containing an antisense message of a gene, or a DNA
sequence encoding a synthetic or modified version of a gene.
[0055] In light of this disclosure, numerous other possible
selectable and/or screenable marker genes, regulatory elements, and
other sequences of interest will be apparent to those of skill in
the art. Therefore, the foregoing discussion is intended to be
exemplary rather than exhaustive.
[0056] After the construction of the plant transformation vector or
construct, said nucleic acid molecule, prepared as a DNA
composition in vitro, is introduced into a suitable host such as E.
coli and mated into another suitable host such as Agrobacterium, or
directly transformed into competent Agrobacterium. These techniques
are well-known to those of skill in the art and have been described
for a number of plant systems including soybean, cotton, and wheat
(See, for example U.S. Pat. Nos. 5,569,834, 5,159,135, and WO
97/48814 herein incorporated by reference in their entirety).
[0057] The present invention encompasses the use of bacterial
strains to introduce one or more genetic components into plants.
Those of skill in the art would recognize the utility of
Agrobacterium-mediated transformation methods. A number of
wild-type and disarmed strains of Agrobacterium tumefaciens and
Agrobacterium rhizogenes harboring Ti or Ri plasmids can be used
for gene transfer into plants. Preferably, the Agrobacterium hosts
contain disarmed Ti and Ri plasmids that do not contain the
oncogenes which cause tumorigenesis or rhizogenesis, respectfully,
which are used as the vectors and contain the genes of interest
that are subsequently introduced into plants. Preferred strains
would include but are not limited to Agrobacterium tumefaciens
strain C58, a nopaline-type strain that is used to mediate the
transfer of DNA into a plant cell, octopine-type strains such as
LBA4404 or succinamopine-type strains e.g., EHA101 or EHA105. The
use of these strains for plant transformation has been reported and
the methods are familiar to those of skill in the art.
[0058] The present invention can be used in any plant
transformation system. Examples of suitable plant targets for the
practice of the present invention would include but are not limited
to alfalfa, barley, canola, corn, cotton, oats, potato, rice, rye,
soybean, sugarbeet, sunflower, sorghum, and wheat. Particularly
preferred dicotyledonous targets would include soybean, cotton,
canola, or sunflower. Particularly preferred monocotyledonous
targets would include cereals such as corn, wheat, and rice.
[0059] The present invention can be used with any transformable
cell or tissue. By transformable as used herein is meant a cell or
tissue that is capable of further propagation to give rise to a
plant. Those of skill in the art recognize that a number of plant
cells or tissues are transformable in which after insertion of
exogenous DNA and appropriate culture conditions the plant cells or
tissues can form into a differentiated plant. Tissue suitable for
these purposes can include but is not limited to immature embryos,
scutellar tissue, suspension cell cultures, callus tissue,
hypocotyl tissue, cotyledons, roots, and leaves. Preferred explants
for dicots include but are not limited to leaf, root, cotyledon,
callus, inflorescence, hypocotyl, and stem. Preferred explants for
monocots include but are not limited to immature embryos,
embryogenic calli, immature inflorescence, root, shoot meristem,
node, nodal explants and cell suspensions.
[0060] The explants can be from a single genotype or from a
combination of genotypes. In a preferred embodiment, superior
explants from plant hybrids can be used as explants. For example, a
fast-growing cell line with a high culture response (higher
frequency of embryogenic callus formation, growth rate, plant
regeneration frequency, etc.) can be generated using hybrid embryos
containing several genotypes. In a preferred embodiment an F1
hybrid or first generation offspring of cross-breeding can be used
as a donor plant and crossed with another genotype. For example,
Pa91 which is an inbred line is crossed with a second inbred line
such as H99 and the resulting F1 hybrid plant is crossed with
inbred A188. Those of skill in the art are aware that heterosis
also referred to herein as "hybrid vigor" occurs when two inbreds
are crossed. The present invention thus encompasses the use of an
explant resulting from a three-way or "triple hybrid" cross,
wherein at least one or more of the inbreds is highly regenerable
and transformable, and the transformation and regeneration
frequency of the triple hybrid explant exceeds the frequencies of
the inbreds individually. Other tissues are also envisioned to have
utility in the practice of the present invention.
[0061] In a preferred embodiment of the present invention, immature
embryos (IEs) of corn, rice, and wheat are used as explants for
Agrobacterium-mediated transformation. In wheat for example,
immature embryos may be isolated from wheat spikelets. The
isolation of wheat immature embryos is also described by Weeks et
al., (1993) and Vasil et al., (1993). Similarly, corn ears are
harvested approximately 8-16 days after pollination and used as a
source of immature embryos. In rice, immature caryopses are
collected from plants after anthesis and immature embryos isolated
from these caryopses are used as explants. The present invention
thus encompasses the use of freshly isolated embryos as described.
In another embodiment a suspension cell culture can be used as
suitable plant material for transformation. In another embodiment a
precultured tissue is used as the target plant material for
transformation. By precultured as used herein is meant culturing
the cells or tissues in an appropriate medium to support plant
tissue growth prior to inoculation with Agrobacterium. The
preculture of the transformable cells or tissue prior to
Agrobacterium inoculation can occur for any length of time, for
example from one day to seven days. Preferably the preculture
period is less than seven days. More preferably the preculture
period is three days or less. Even more preferably, the preculture
of the transformable cells or tissues is from 18-28 hours.
[0062] Any suitable plant culture medium can be used for the
preculture. Examples of suitable media for preculture would include
but are not limited to MS-based media (Murashige and Skoog, 1962)
or N6-based media (Chu et al., 1975) supplemented with additional
plant growth regulators including but not limited to auxins such as
picloram (4-amino-3,5,6-trichloropicolinic acid), 2,4-D
(2,4-dichlorophenoxyacetic acid) and dicamba (3,6-dichloroanisic
acid), cytokinins such as BAP (6-benzylaminopurine) and kinetin,
and gibberellins. Other media additives can include but are not
limited to amino acids, macroelements, iron, microelements,
vitamins and organics, carbohydrates, undefined media components
such as casein hydrolysates, an appropriate gelling agent such as a
form of agar, such as a low melting point agarose or Gelrite if
desired. 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 (Murashige and Skoog, 1962), N6
(Chu et al., 1975), Linsmaier and Skoog (Linsmaier and Skoog,
1965), Uchimiya and Murashige (Uchimiya and Murashige, 1974),
Gamborg's media (Gamborg et al., 1968), D medium (Duncan et al.,
1985), McCown's Woody plant media (McCown and Loyd, 1981), Nitsch
and Nitsch (Nitsch and Nitsch, 1969), and Schenk and Hildebrandt
(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 that can be optimized for the particular
target crop of interest.
[0063] Once the transformable plant tissue is isolated, the next
step of the method is introducing the genetic components into the
plant tissue. This process is also referred to herein as
"transformation." The plant cells are transformed and each
independently transformed plant cell is selected. The independent
transformants are referred to as transgenic events. A number of
methods have been reported and can be used to insert genetic
components into transformable plant tissue.
[0064] Methods for transforming dicots, primarily by use of
Agrobacterium tumefaciens, and obtaining transgenic plants have
been published for a number of crops including cotton (U.S. Pat.
No. 5,064,863; U.S. Pat. No. 5,159,135; U.S. Pat. No. 5,518,908, WO
97/43430), soybean (U.S. Pat. No. 5,569,834; U.S. Pat. No.
5,416,011; McCabe et al. (1988); Christou et al. (1988), Brassica
(U.S. Pat. No. 5,463,174), peanut (Cheng et al. (1996); De Kathen
and Jacobsen (1990)).
[0065] Transformation of monocots using electroporation, particle
bombardment, and Agrobacterium have also been reported.
Transformation and plant regeneration have been achieved in
asparagus (Bytebier et al. (1987)), barley (Wan and Lemaux (1994),
Tingay et al., (1997)' maize (Rhodes et al. (1988); Ishida et al.
(1996); Gordon-Kamm et al. (1990); Fromm et al. (1990); Koziel et
al. (1993); Armstrong et al. (1995), oat (Somers et al. (1992)),
rice (Toriyama et al. (1988); Zhang and Wu (1988); Zhang et al.
(1988); Battraw and Hall (1990); Christou et al. (1991); Hiei et
al., 1994; Park et al. (1996)), sugarcane (Bower and Birch (1992),
Arencibia et al., 1998, tall fescue (Wang et al. (1992)), and wheat
(Vasil et al. (1992); Weeks et al. (1993), Cheng et al.,
1997)).
[0066] The present invention utilizes Agrobacterium-mediated
transformation. One advantage of the present invention is that the
presence of additional virulence genes is not required.
Transformation was achieved in all plant systems tested. The fact
that a super binary vector may not be necessary provides added
utility, whereas it has been shown to be essential for achieving
high transformation in another reported maize system (Ishida et
al., 1996).
[0067] In a preferred embodiment, a transformable tissue is
inoculated with Agrobacterium in the presence of an growth
inhibiting agent. By growth inhibiting agent as used herein is
meant any agent that is capable of stressing, suppressing,
limiting, or inhibiting bacterial cell growth. Preferably, the
growth inhibiting agent inhibits Agrobacterium cell growth. More
preferably the growth inhibiting agent inhibits Agrobacterium
tumefaciens cell growth and reduces the T-DNA transfer process. The
agents referred to herein may be chemical or biological agents. Any
number of methods or agents to suppress or inhibit Agrobacterium
growth are envisioned. An agent that is toxic (bacteriostatic or
bacteriocidal) to the Agrobacterium and less toxic to the plant
cells can be included in the stages in the transformation process
up to the selection step. Preferably one or more growth inhibiting
agents are included with Agrobacterium at a concentration that is
effective in stressing, suppressing, or inhibiting Agrobacterium
growth yet remaining neutral or positive with respect to plant cell
growth. Accordingly, depending on the plant system and media
components, the effective concentration and duration of inclusion
of the growth inhibiting agent(s) can vary and can be optimized.
For example, any agent can be tested for the effect of said agent
on Agrobacterium cell growth by any number of methods including but
not limited to testing the agent in different concentrations, in
different culture conditions, and in different plant systems using
methods known to those of skill in the art. These stages for
including one or more growth inhibiting agents would include any
stage in a transformation process during which Agrobacterium and a
plant cell are together and during which T-DNA transfer can occur.
The particularly preferred transformation stages would include
inoculation, wounding, and co-culture steps, including prolonged
co-culture during in planta transformation methods (Bechtold et
al., 1993; Clough and Bent, 1998). T-DNA transfer is a biological
process and inclusion of such an growth inhibiting agent during the
inoculation, wounding, co-cultivation, and/or infiltration steps
can also inhibit the T-DNA processing and transfer. The growth
inhibiting agent can be present either singly or in combination
with other growth inhibiting agents. Examples of suitable growth
inhibiting agents include but are not limited to antibiotics such
as amphotericinB, carbenicillin, cefotaxime, chloramphenicol,
cycloheximide, erythromycin, gentamicin A, sulphate, geneticin,
hygromycin B, hydroxyquinoline, kanamycin, methotrexate, naladixic
acid, neomycin sulphate, nystatin, paromomycin, penicillin,
pentachloronitrobenzene, rifampicin, streptomycin, sulphonamide,
tetracycline, trimethoprim, thiabendazole, ticarcillin, vancomycin,
spectinomycin, compounds containing heavy metals such as silver
nitrate silver thiosulfate, silver nitrite, silver di-thionate,
silver stearate, silver selenate, silver salicylate, silver
oxalate, silver phosphate, silver metaphosphate, silver
orthophosphate, silver orthophosphate mono-H, silver carbonate,
silver propionate, silver acetate, silver citrate, silver laurate,
silver levunilate, silver pyrophosphate or other silver-containing
compounds, other chemicals such as compounds containing potassium,
manganese, or cadmium, proteins, nucleotides, and cell extracts,
cell exudates, secondary metabolites, sulfa-drugs, and growth
regulators. A derivative as used herein refers to other forms of
the growth inhibiting agent including but not limited to a salt
derivative, an anhydrous derivative, or a hydrated derivative that
are capable of inhibiting Agrobacterium growth. Particularly
preferred growth inhibiting agents would include silver nitrate,
silver thiosulfate, and penicillins such as carbenicillin,
ampicillin, and cloxacillin, cephalosporins such as cefotaxime and
cefoxitin, or a combination antibiotic such as a penicillin plus
clavulanic acid such as augmentin and timentin. The growth
inhibiting agents can be "included" during the inoculation and
post-inoculation stages by a number of ways, depending on the
nature of the agent. Chemical agents for example can be included in
the culture media by addition from a stock solution, or can be
added in solid form. The agent may be adhered to a support matrix
such as a piece of filter paper and placed on semi-solid, a solid
support, or liquid media. The agent can also be added to a vacuum
infiltration medium or during the process of sonication-assisted
Agrobacterium-mediated transformation (Trick et al., 1997).
[0068] In another embodiment, a nucleic acid sequence such as an
intron can be included in the selectable marker gene to slow down
or inhibit Agrobacterium cell growth during the co-cultivation and
transformation process. It has been reported that a promoter of
microbial origin e.g. 35S, NOS, etc., can regulate expression of
genes in Agrobacterium cells. An intron-containing antibiotic
marker gene can be used to inhibit Agrobacterium cells by using a
differential selection strategy, e.g. nptII (conferring resistance
to kanamycin), aphIV (conferring resistance to hygromycin B), acC3
and aacC4(conferring resistance to gentamycin) or aadA (conferring
resistance to spectinomycin and streptomycin). For example, plant
cells are rarely sensitive to kanamycin at a concentration of 25
mg/L but the same concentration is lethal to Agrobacterium
cells.
[0069] In another embodiment a growth inhibiting agent is a
nucleotide sequence that inhibits Agrobacterium cell growth and
inhibits T-DNA processing, transfer, and integration. This can be
achieved by introducing and regulating the expression of a sense or
antisense gene(s) in the Agrobacterium cells. Selective regulation
of such a gene or genes(s) can allow the manipulation of T-DNA
mediated gene delivery. Suitable genes would include but are not
limited to metabolic genes involved in pathways for carbohydrate
metabolism.
[0070] The growth inhibiting agent can be added in an amount
sufficient to achieve a desired effect on Agrobacterium growth. The
effective range of the agent can be manipulated to determine the
optimal concentration of agent. The concentration of the growth
inhibiting agent can vary depending on culture conditions including
but not limited to media components and the plant system used. For
example, different media components can interact with the
inhibiting agent(s) and affect the amount of agent needed under
certain culture conditions for a particular plant tissue system. In
one embodiment, one or more growth suppressing agents can be
combined and included either together, or in different stages of
the transformation process. Preferably the presence of the agent(s)
with Agrobacterium is effective such that the density of the
Agrobacterium does not increase in the presence of the agent. More
preferably, the presence of the agent(s) has a negative effect on
Agrobacterium growth and has a neutral or positive effect on plant
growth.
[0071] In further embodiments of the invention, the growth
inhibiting agent may be included only in the inoculation step, only
in the co-culture step, or in both the inoculation and co-culture
steps.
[0072] Those of skill in the art are aware of the typical steps in
the plant transformation process. The Agrobacterium can be prepared
either by inoculating a liquid such as Luria Burtani (LB) media
directly from a glycerol or streaking the Agrobacterium onto a
solidified media from a glycerol, allowing the bacteria to grow
under the appropriate selective conditions, generally from about
26.degree. C.-30.degree. C., more preferably about 28.degree. C.,
and taking a single colony from the plate and inoculating a liquid
culture medium containing the selective agents. Alternatively a
loopful or slurry of Agrobacterium can be taken from the plate and
resuspended in liquid and used for inoculation. Those of skill in
the art are familiar with procedures for growth and suitable
culture conditions for Agrobacterium as well as subsequent
inoculation procedures. The density of the Agrobacterium culture
used for inoculation and the ratio of Agrobacterium cells to
explant can vary from one system to the next, and therefore
optimization of these parameters for any transformation method is
expected.
[0073] Typically, an Agrobacterium culture is inoculated from a
streaked plate or glycerol stock and is grown overnight and the
bacterial cells are washed and resuspended in a culture medium
suitable for inoculation of the explant. Suitable inoculation media
for the present invention include, but are not limited {fraction
(1/2)} MS PL or {fraction (1/2)} MS VI (TABLE 3). Preferably, the
inoculation media is supplemented with the growth inhibition agent.
The range and concentration of the growth inhibition agent can vary
and depends of the agent and plant system. For the present
invention silver nitrate, silver thiosulfate, or carbenicillin are
the preferred growth inhibition agents. The growth inhibiting agent
is added in the amount necessary to achieve the desired effect.
Silver nitrate is preferably used in the inoculation media at a
concentration of about 1 .mu.M (micromolar) to 1 mM (millimolar),
more preferably 5 .mu.M-100 .mu.M. the concentration of
carbenicillin used in the inoculation media is about 5 mg/L to 100
mg/L, more preferably about 50 mg/L. An Agrobacterium virulence
inducer such as acetosyringone can also be added to the inoculation
media.
[0074] In a preferred embodiment, the Agrobacterium used for
inoculation are pre-induced in a medium such as a buffered media
with appropriate salts containing acetosyringone, a carbohydrate,
and selective antibiotics. In a preferred embodiment, the
Agrobacterium cultures used for transformation are pre-induced by
culturing at about 28.degree. C. in AB-glucose minimal medium
(Chilton et al., 1974; Lichtenstein and Draper, 1986) supplemented
with acetosyringone at about 200M and glucose at about 2%. The
concentration of selective antibiotics for the Agrobacterium in the
pre-induction medium is about half the concentation normally used
selection. The density of the Agrobacterium cells used is about
10.sup.7-10.sup.10 cfu/ml of Agrobacterium. More preferably, the
density of Agrobacterium cells used is about
5.times.10.sup.8-4.times.10.sup.9. Prior to inoculation the
Agrobacterium can be washed in a suitable media such as {fraction
(1/2)} MS.
[0075] The next stage of the transformation process is the
inoculation. In this stage the explants and Agrobacterium cell
suspensions are mixed together. The mixture of Agrobacterium and
explant(s) can also occur prior to or after a wounding step. By
wounding as used herein is meant any method to disrupt the plant
cells thereby allowing the Agrobacterium to interact with the plant
cells. Those of skill in the art are aware of the numerous methods
for wounding. These methods would include but are not limited to
particle bombardment of plant tissues, sonicating, vacuum
infiltrating, shearing, piercing, poking, cutting, or tearing plant
tissues with a scalpel, needle or other device. The duration and
condition of the inoculation and Agrobacterium cell density will
vary depending on the plant transformation system. The inoculation
is generally performed at a temperature of about 15.degree.
C.-30.degree. C., preferably 23.degree. C.-28.degree. C. from less
than one minute to about 3 hours. The inoculation can also be done
using a vacuum infiltration system.
[0076] Any Agrobacterium growth inhibiting agent or combination of
agents can be included in the inoculation medium. For the present
invention examples of growth inhibiting agents such as silver
nitrate, silver thiosulfate, or carbenicillin are included in an
MS-based inoculation medium. The concentration of silver nitrate or
silver thiosulfate in the inoculation media can range from 1 .mu.M
to 1 mM, more preferably from 5 .mu.M to 100 .mu.M, even more
preferably, from about 10 .mu.M to 50 .mu.M, most preferably about
20 .mu.M. The concentration of carbenicillin the inoculation medium
is from about 5 mg/L to 1000 mg/L, more preferably, about 10 mg/L
to 50 mg/L, even more preferably, about 50 mg/L.
[0077] After inoculation any excess Agrobacterium suspension can be
removed and the Agrobacterium and target plant material are
co-cultured. The co-culture refers to the time post-inoculation and
prior to transfer to a delay or selection medium. Any number of
plant tissue culture media can be used for the co-culture step. For
the present invention a reduced salt media such as {fraction (1/2)}
MS-based co-culture media (TABLE 4) is used and the media lacks
complex media additives including but not limited to undefined
additives such as casein hydolysate, and B5 vitamins and organic
additives. Plant tissues after inoculation with Agrobacterium can
be cultured in a liquid media. More preferably, plant tissues after
inoculation with Agrobacterium are cultured on a semi-solid culture
medium solidified with a gelling agent such as agarose, more
preferably a low EEO agarose. The co-culture duration is from about
one hour to 72 hours, preferably less than 36 hours, more
preferably about 6 hours to 35 hours. The co-culture media can
contain one or more Agrobacterium growth inhibiting agent(s) or
combination of growth inhibiting agents. Preferably the co-culture
media contains an Agrobacterium growth inhibiting agent such as
silver nitrate, silver thiosulfate, or carbenicillin. The
concentration of silver nitrate or silver thiosulfate is preferably
about 1 .mu.M to 1 mM, more preferably about 5 .mu.M to 100 .mu.M,
even more preferably about 10 .mu.M to 50 .mu.M, most preferably
about 20 .mu.M. The concentration of carbenicillin in the
co-culture medium is preferably about 5 mg/L to 100 mg/L more
preferably 10 mg/L to 50 mg/L, even more preferably, about 50 mg/L.
The co-culture is typically performed for about one to three days
more preferably for less than 24 hours at a temperature of about
18.degree. C.-30.degree. C., more preferably about 23.degree.
C.-25.degree. C. The co-culture can be performed in the light or in
light-limiting conditions. Preferably, the co-culture is performed
in light-limiting conditions. By light-limiting conditions as used
herein is meant any conditions which limit light during the
co-culture period including but not limited to covering a culture
dish containing plant/Agrobacterium mixture with a cloth, foil, or
placing the culture dishes in a black bag, or placing the cultured
cells in a dark room. Lighting conditions can be optimized for each
plant system as is known to those of skill in the art.
[0078] After co-culture with Agrobacterium, the explants can be
placed directly onto selective media. The explants can be
sub-cultured onto selective media in successive steps or stages.
For example, the first selective media can contain a low amount of
selective agent, and the next sub-culture can contain a higher
concentration of selective agent or vice versa. The explants can
also be placed directly on a fixed concentration of selective
agent. Alternatively, after co-culture with Agrobacterium, the
explants can be placed on media without the selective agent. Those
of skill in the art are aware of the numerous modifications in
selective regimes, media, and growth conditions that can be varied
depending on the plant system and the selective agent. In the
preferred embodiment, after incubation on non-selective media
containing the antibiotics to inhibit Agrobacterium growth without
selective agents, the explants are cultured on selective growth
media. Typical selective agents include but are not limited to
antibiotics such as geneticin (G418), kanamycin, paromomycin or
other chemicals such as glyphosate. Additional appropriate media
components can be added to the selection or delay medium to inhibit
Agrobacterium growth. Such media components can include, but are
not limited to antibiotics such as carbenicillin or cefotaxime.
[0079] After the co-culture step to inhibit Agrobacterium growth,
and preferably before the explants are placed on selective or delay
media, they can be analyzed for efficiency of DNA delivery by a
transient assay that can be used to detect the presence of one or
more gene(s) contained on the transformation vector, including, but
not limited to a screenable marker gene such as the gene that codes
for .beta.-glucuronidase (GUS). The total number of blue spots
(indicating GUS expression) for a selected number of explants is
used as a positive correlation of DNA transfer efficiency. The
efficiency of T-DNA delivery and the effect of Agrobacterium growth
inhibiting agents on T-DNA delivery and a prediction of
transformation efficiencies can be tested in transient analyses as
described. A reduction in the T-DNA transfer process can result in
a decrease in copy number and complexity of integration as complex
integration patterns can originate from co-integration of separate
T-DNAs (DeNeve et al., 1997). The effect of Agrobacterium growth
inhibiting agents on reducing copy number by influencing T-DNA
transfer and transformation efficiency can be tested by transient
analyses and more preferably in stably transformed plants. Any
number of methods are suitable for plant analyses including but not
limited to histochemical assays, biological assays, and molecular
analyses.
[0080] In a preferred embodiment additional experiments can be
performed to assess the effect of growth inhibiting agents on
Agrobacterium cells and plant growth for any plant transformation
system. For example, Agrobacterium growth can be monitored in the
presence and absence of one or more growth inhibiting agents at
different concentrations and at different timepoints in the
transformation process. In one embodiment, the effect of a growth
inhibiting agent on Agrobacterium can be monitored by quantitating
the recovery of Agrobacterium after a step in the process in a
comparison with and without the growth inhibiting agent(s).
[0081] In another embodiment, plant cells can be infected with a
wild-type tumor-inducing Agrobacterium strain and the effect of one
or more growth inhibiting agents on tumor formation can be assessed
by evaluating tumor formation in the presence or absence of the
agent(s). T-DNA transfer can be monitored on the basis of a
transient assay including but not limited to an assay for
-glucuronidase (GUS) assay (Jefferson, R. A., 1987).
[0082] The cultures are subsequently transferred to a media
suitable for the recovery of transformed plantlets. Those of skill
in the art are aware of the number of methods to recover
transformed plants. 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.
[0083] The transformants produced are subsequently analyzed to
determine the presence or absence of a particular nucleic acid of
interest contained on the transformation vector. Molecular analyses
can include but is not limited to Southern blots (Southern, 1975),
or PCR (polymerase chain reaction) 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 and have been reported
(See for example, Sambrook et. al., Molecular Cloning, A Laboratory
Manual, 1989).
[0084] Those of skill in the art will appreciate the many
advantages of the methods and compositions provided by the present
invention. The following examples are included to demonstrate the
preferred 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
inventors to function well in the practice of the invention, and
thus can be considered to constitute preferred modes for its
practice. 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 that are disclosed and still obtain a like
or similar result without departing from the spirit and scope of
the invention.
EXAMPLES
Example 1
[0085] Bacterial Strains and Plasmids
[0086] The Agrobacterium strains and binary plasmid vectors used
are listed in Table 1. Plasmid vectors were constructed using
standard molecular biological techniques known to one of ordinary
skill in the art. Briefly, the plant transformation vectors
described herein comprise one or more nucleic acid sequences
including but not limited to one or more T-DNA border sequences
(right border, RB; left border, LB) to promote the transfer of
nucleic acid molecules into the plant genome, replication elements,
a selectable marker and one or more gene(s) of interest. The
plasmid vectors tested are shown in FIG. 1-FIG. 5).
[0087] A brief description of the plasmids is as follows: the e35S
promoter is a modification of the 35S promoter derived from the 35S
RNA of cauliflower mosaic virus (CaMV) that contains a duplication
of the -90 to -300 region; the nos promoter is from Agrobacterium
tumefaciens pTiT37. the GUS gene is the .beta.-glucuronidase coding
sequence from E. coli modified to have a Nco site at the start
codon; ST-LS1*NT is the intron from Solanum tuberosum; the nptII
gene (kan) codes for neomycin phosphotransferase; the nos 3' region
contains downstream untranslated sequence and the poly A signal for
the NOS gene of Agrobacterium tumefaciens pTiT37; ori-V is the
vegetative origin of replication; ori-322 is the minimum known
sequence for a function origin of replication; the CP4 gene is the
coding sequence for EPSP synthase, (confers tolerance to the
glyphosate herbicide); GFP is a modified coding sequence for green
fluorescent protein, The selectable (nptII) and reporter genes
(uidA) are driven by an enhanced 35S promoter (E35S; fig.) followed
by an untranslated hsp 70 intron (Rochester et al., 1986); The uidA
has an additional intron within the coding sequence to minimize
bacterial expression (Vancannyet et al., 1990);
[0088] the bar gene confers resistance to the herbicide bialaphos;
the gent gene confers resistance to gentamycin; P-ract1 and
ract1intron refer to the rice actin promoter and rice actin intron
respectively.
[0089] Binary plasmids were introduced into different Agrobacterium
strains through electroporation using Bio-Rad Gene Pulser, operated
at 2.5 kv and 400 Ohms. Transconjugants were selected on semi-solid
Luria-Bertani medium, LB using appropriate antibiotics.
1TABLE 1 Agrobacterium strains and plasmids Strain or plasmid
Relevant characteristics Reference/Figure A136 C58 cured of pTiC58
Watson et. al. 1975 ABI C58 with pMP90RK Koncz et al. 1986 A281
A136 (pTiBo542) Sciaky et. al. 1978 (succinamopine-type) EHA 101
Disarmed A281 Hood et al. 1986 pMON 30100 derivative of pPZP100
Hajdukiewicz et al. 1994, FIG. 1 pMON 18365 ABI compatible binary
vector pMON25457 derivative of pMON30100 pMON25492 CP4 linear
vector pMON32092 derivative of pMON30100
Example 2
[0090] Pre-Induction of Agrobacterium
[0091] Agrobacterium cultures used for transformation are
pre-induced (except as otherwise indicated) by acetosyringone (200
.mu.M) and glucose (2%) in AB based induction medium. The procedure
followed was as follows:
[0092] 1.sup.st Step:
[0093] A loopful of bacterial colonies were picked from a freshly
plated plate and grown at 28.degree. C. in 50 mls of LB medium
containing appropriate antibiotics for 15-24 h. The optical density
of the bacterial culture at the end of the culture period was
.about.1.4 at A.sub.660.
[0094] 2.sup.nd Step:
[0095] A 10 ml aliquot of these cells were transferred into a 50
mls of fresh LB with appropriate antibiotics and grown for an
another period of 6-8 h (to an optical density of .about.1.2).
[0096] 3.sup.rd Step:
[0097] Agrobacterium cells were centrifuged at 4.degree. C. for 10
min at 3250 g and the pellet was resuspended in the pre-induction
medium to a final optical density of 0.2 at A.sub.660 and incubated
at 28.degree. C. for 12-15 h.
[0098] 4.sup.th Step:
[0099] Prior to use for transformation, the Agrobacterium cells
were centrifuged at 4.degree. C. for 10 min at 3250 g. After
decanting the supernatant, the pellet was resuspended in {fraction
(1/2)} MS wash medium (at least 100 ml of {fraction (1/2)} MS wash
medium for 1L Agrobacterium cultures was used), aliquoted into 50
ml centrifuge tubes, centrifuged cells at 4.degree. C. for 10 min
at 3250 g, removed the supernatant and stored the tubes with
pellets in ice till use (the Agrobacterium cells can be stored on
ice up to 4 hr).
[0100] Reagents are commercially available and can be purchased
from a number of suppliers (see, for example Sigma Chemical Co.,
St. Louis, Mo.).
2TABLE 2 Pre-induction medium*.sup.1 100 mM MES (pH 5.4) 1X AB
salts 0.5 mM NaH.sub.2PO.sub.4 2% Glucose acetosyringone 200 .mu.M
*The concentration of antibiotics in the pre-induction medium are
0.5.times. of the concentration used in LB medium. For example, the
antibiotic concentrations used for selection of EHA101(pMON25457)
grown in LB were (in g/ml) Kan.sub.100 plus Gent .sub.100, and in
the induction medium the level used is 50 mg/L Kanamycin and 50
mg/L Gentamycin. For C58-ABI strains selection concentrations used
are: 100 mg/L Kanamycin, 100 mg/L Spectinomycin, 100 mg/L
Streptomycin, and 25 mg/L # Chloramphenicol in the LB medium and 50
mg/L Kanamycin, 50 mg/L Spectinomycin, 50 mg/L Streptomycin, and 25
mg/L Chloramphenicol in the induction medium. .sup.1final
concentration
Example 3
[0101] Explant Preparation
[0102] Several explants were used in this study:
[0103] 1) Young kalanchoe plants were grown in the green house. The
leaves of this plant were used for the transformation
experiment.
[0104] 2) A very fine suspension cell line of Zea mays L. of Black
Mexican Sweet (BMS) (BMS; Sheridan, 1975; Chourey and Zurawski,
1981), with maximum of .about.100 cells/clump size and a doubling
time of approximately two days was used with the experiments with
BMS. BMS cells were maintained in a modified liquid Murashige and
Skoog medium, MS-BMS (Table 9). Suspension culture were maintained
at 28.degree. C. in the dark on a horizontal shaker at 150 rpm and
were sub-cultured at 2 day intervals by diluting 25 mls of cell
suspensions with 50 mls of fresh medium.
[0105] 3) Immature embryo: Immature embryos from several crops e.g.
corn, rice and wheat were used.
[0106] Corn
[0107] Several genotypes of corn were used in this study including
H99, (H99 X Pa91)A188, H99 .times.A188, LH198 .times.Hi-II. Ears
containing immature embryos were harvested approximately 10 days
after pollination and kept refrigerated at 4.degree. C. until use
(up to 5 days post-harvest). The preferred embryo size for this
method of transformation is .about.1.5-2.5 mm for the hybrid
(Pa91.times.H99) A188. This size is usually achieved 10 days after
pollination inside the green house. with the following growth
conditions with an average temperature of 87.degree. F., day length
of 14 hours with supplemental lighting supplied by GE 1000 Watt
High Pressure Sodium lamps.
[0108] Rice
[0109] A California variety M202 was used and is publicly
available. Stock plants were grown in a greenhouse with an average
temperature of 78.degree. F. day/70.degree. F. night, day length 14
hours with supplemental lighting supplied by GE 1000 Watt High
Pressure Sodium lamps. Immature caryopses were collected from
plants 7-12-d after anthesis. IEs were dissected aseptically and
either used directly for transformation or pre-cultured on MS
callus induction medium (MS1) before inoculation. All cultures were
incubated at the temp. of 23-25.degree. C.
[0110] Wheat
[0111] A spring wheat Triticum aestivum cv. Bobwhite was used.
Stock plants were grown in an environmentally controlled chamber
with 16-h photoperiod at 800 .mu.mol m-2 s-1 provided by
high-intensity discharge Sylvania lights (GTE Products Corp.,
Manchester, N.H.). The day and night temperatures were
18/16.degree. C. Immature caryopses were collected from plants 14-d
after anthesis. IEs were dissected aseptically and directly used
for transformation or pre-cultured on MS callus induction medium
before inoculation. In other cases, cultures were incubated at
23-25.degree. C.
[0112] Other Explants
[0113] Any other explants not described in this section are
described in detail under the specific EXAMPLES.
Example 4
[0114] Inoculation
[0115] The duration and condition of the inoculation and
Agrobacterium cell density varied throughout the course of this
invention and are described in detail in the specific EXAMPLES.
[0116] The following method of inoculation applies to all explants
other than BMS suspension cells. The procedure on BMS cell
suspension transformation is described in TABLE 8. The
Agrobacterium suspension was resuspended to a desired optical
density (OD A.sub.660 1.0=10.sup.9 cfu/ml) with {fraction (1/2 )}
MS PL medium supplemented with acetosyringone (200 .mu.M) and other
bacteriocidal chemicals (as necessary). Three mls of this
Agrobacterium suspension culture was added into a 6-well plate
(Coster non treated 6-well plates, Corning Inc., Acton, Mass.). IEs
were isolated for 10-15 minutes directly into each well (if using
freshly isolated IEs as explants) and inoculation was performed for
an additional 15 minutes after the isolation period.
[0117] After the inoculation period most of the Agrobacterium
suspension was gently removed using a sterile transfer pipette.
Embryos were gently collected with a sterile spatula and .about.50
embryos were transferred to a single co-culture plate. During
co-culture the plates containing embryos were incubated at
23.degree. C. for 1-3 days. During the transformation process,
exposure of co-culture plates to light was minimized by covering
the plates with foil or a dark cloth.
3TABLE 3 Inoculation media Final Concentration: 1/2 MS VI
inoculation medium* MS salts 2.2 g/L (Gibco) 1X MS vitamins 1 ml of
1000X stock Proline 115 mg/L Glucose 10 g/L Sucrose 20 g/L
Acetosyringone 200 .mu.M (200 .mu.l/l of 1M stock) pH 5.4 with KOH
Filter sterilize Add acetosyringone 200 .mu.M (fresh) to the medium
prior to using. *Used for vacuum infiltration of BMS and IEs and
for washing Agrobacterium cells 1/2 MS PL inoculation medium* MS
salt 2.2 g/L (Gibco) 1X MS vitamins 1 ml of 1000X stock Proline 115
mg/L Glucose 36 g/L Sucrose 68.5 g/L Acetosyringone 200 .mu.M (200
.mu.l/l of 1M stock) pH 5.2 with KOH Filter sterilize Add
acetosyringone 200 .mu.M to the medium prior to using. *Used for
stable transformation of all explants except for BMS unless
otherwise indicated.
Example 5
[0118] Co-Culture
[0119] The conditions for co-culture (time period post-inoculation
and prior to transfer of explants to delay, prolonged co-culture
(kalanchoe leaves) or selection medium) varied depending on the
plant system. The various media used are outlined below in the
following tables.
4TABLE 4*.sup.1 Co-culture Medium 1/2 MS CC MS salt 2.2 g/L (Gibco)
1X MS vitamins 1 ml of 1000X stock Thiamine HCl 0.5 mg/L Proline
115 mg/L Glucose 10 g/L Sucrose 20 g/L 2,4-D 3 mg/L Low EEO agarose
5.5 g/L Acetosyringone 200 .mu.M (200 .mu.l/l of 1M stock)
Bacteriocidal additives described in specific EXAMPLES Made 2X
stock, pH 5.2 with KOH, filter sterilized Added acetosyringone (200
.mu.M) & growth inhibiting agents to the medium prior to
pouring plates. *Used for stable transformation of all explants of
all crops except for BMS and unless otherwise indicated.
.sup.1Final concentration
[0120]
5TABLE 5 Co-culture Medium 1/2 MS BMS*.sup.1 MS salts 2.2 g/L
(Gibco) 1X MS vitamins 1 ml of 1000X stock Thiamine HCl 0.5 mg/L
Asparagine 150 mg/L L-Proline 115 mg/L Glucose 10 g/L Sucrose 20
g/L 2,4-D 3 mg/L Acetosyringone 200 .mu.M (200 .mu.l/l of 1M stock)
pH 5.2 with KOH, filter sterilize 200 .mu.M Acetosyringone is added
to the medium (fresh). *Used for stable transformation of all
explants of all crops except for BMS and unless otherwise
mentioned. .sup.1Final concentration
Example 6
[0121] Transformation Methods for Corn, Wheat, and Rice (Various
Selectable Markers)
6TABLE 6 Method for Agrobacterium-Mediated Corn Transformation 1.
Inoculation: Inoculation duration for 15 minutes--3 hours with or
without vacuum. 2. Co-culture (1-3 days): Duration of co-culture at
23.degree. C. on 1/2MSC (Table 4). 3. Delay (3-7 days): Culture on
D medium (Duncan et al., 1985) supplemented with 3 mg/L 2,4-D,
250-500 mg/L Cefatoxime plus 20 .mu.M AgNO.sub.3). 4. 1.sup.st
selection (2 wks)*: D medium supplemented with 500 mg/L
Carbenicillin plus 50 mg/L {aromomycin. At this stage coleoptiles
were removed if present and sub-culturing was not necessary)
.about.25 embryos/plate 5. 2.sup.nd selection (2-3 wks): 15A1A (D
medium) plus 375 mg/L Carbenicillin plus 100 mg/L Paromomycin. At
this stage, sub-culturing was necessary. The size of transformed
sectors were usually .about.2 mm and a positive embryos had only
few sectors. .about.17 embryos/plate 6. 3.sup.rd selection (2-3
wks): D medium supplemented with 250 mg/L Carbenicillin plus 200
mg/L Paromomycin. At this stage sub-culturing was necessary. 7.
1.sup.st regeneration (5-7 d): Transferred resistant pieces to the
regeneration medium supple- mented with 3.5 mg/L BA and 100 mg/L
Carbenicillin and incubated in the dark. 8. 2.sup.nd regeneration
(3 weeks): MSOD with 100 mg/L Carbenicillin and 50 mg/L Paromomycin
*For non-hybrid embryos <50 mg/L Paromomycin e.g. H99 25 mg/L
Paromomycin for 2 weeks
[0122]
7TABLE 7 Supplemental Components in Basic Media Used for Corn
Transformation Using nptII* Components 1/2MS CC.sup.2 Delay.sup.1
Selection.sup.1 Reg.sup.1 MSOD.sup.2 2,4-D (mg/L) 3.0 -- -- -- --
BAP (mg/L) -- -- -- 3.5 -- Dicamba (mM) -- 15 15 -- -- Glucose
(g/L) 10 10 10 10 10 Sucrose (g/L) 20 20 20 20 -- Maltose (g/L) --
-- -- -- 20 L-Asparagine (15 mg/ml stock) -- -- -- -- 10 ml
Myo-Inositol (g/L) -- -- -- -- 0.1 MS mod. Vitamins (1000X).sup.4
-- -- -- 1 ml L-Proline 1.0 12 12 12 -- (mM) gelling agent
(g/L).sup.3 5.5 (A) 7.0 (P) 7.0 (P) 7.0 (P) 5.0 (G) AgNO3 (M)**
AgNO3 amount added as indicated in Examples Carbenicillin -- -- 500
375 250 (mg/L) Cefatoxime -- 250/500 -- -- -- (mg/L) pH 5.4 5.8 5.8
5.8 5.8 .sup.1Media contained basal salts and vitamins (Duncan et
al., 1985) .sup.2Media contained basal salts and vitamins) from
(Murashige and Skoog) *All media components were filter sterilized
and added to the medium after autoclaving. .sup.3Low-EEO Agarose
(A) or Phytagar .TM. (P) or Agargel .TM. (G) all commercially
available (see for example Sigma Chemical, St. Louis, MO).
.sup.4Table 9.
[0123]
8TABLE 8 Protocol for Transforming Black Mexican Sweet Suspension
Cells 1. Rapidly growing BMS suspension cells were sub-cultured at
an interval of 2 days by taking 25 mls of cell suspensions and
diluting the suspension with 50 mls of fresh medium (MS-BMS, Table
3). 2. 10 ml of cells added (= 1 ml packed cell volume) into a six
well culture plates (Corning Coster nontreated 6-well plates) and
removed 9.5 mls of medium. 3. Added 3 mls of pre-induced
Agrobacterium suspensions (Agrobacterium preparation) and gently
suspended BMS cells in Agrobacterium suspension 4. Inoculated 3
hours under vacuum 5. Removed all Agrobacterium suspension 6. Added
10 mls of wash medium 7. Plated half of the suspension cells (5.5
mls) of cell suspension from each well onto a filter paper (Baxter
5.5 catalog #F2217-55, Baxter Scientific) using a buchner funnel
and vacuum 8. Transferred each filter paper with cells to
co-culture medium (1/2 BMS co-culture supplemented with 200 .mu.M
acetosyringone). Co-culture plates were prepared by placing 2
filter papers (Baxter) soaked with 3.5 mls of co-culture media in
20 .times. 60 mm plates. Co-culture was performed for 1-3 days at
23.degree. C. in the dark. 9. At the end of co-culture period, the
filter paper with cells were washed with 25 mls of MS-BMS liquid
plus 750 mg/L Carbenicillin under gentle vacuum using a buchner
funnel. Transient analyses were performed at this stage. For the
recovery of stable transformants, the entire filter paper with
cells was transferred to the selection medium. 10. Each filter
paper with plated cells was transferred onto 1.sup.st selection
medium (MS- BMS) supplemented with 200 mg/L Kanamycin and 750 mg/L
Carbenicillin supplemented with 10% conditioning medium (prepared
from one day old BMS suspension culture by taking cell free
supernatant). Selection plates were prepared by putting 2 filter
papers (7.0 cm Baxter, Cat# F2217-70) soaked with 5 mls of 1.sup.st
selection media. Plates were sealed with parafilm and the culture
was performed for 5 days at 28.degree. C. 11. Each filter paper
with cells was transferred onto semi-solid MS-BMS medium containing
20 mg/L Paromomycin and 750 mg/L Carbenicillin at 2 week intervals.
12. The efficiency of transformation was scored by counting GUS
positive colonies 5 weeks after co-culture.
[0124]
9TABLE 9 Supplemental Components in MS Modified Medium (MS-BMS) for
BMS Suspension Culture and Transformation.sup.1,2 Components
Amount/Liter 2,4-D (mg/L) 2.0 Sucrose (g/L) 20 L-Asparagine (15
mg/ml stock) 10 ml Myo-Inositol (g/L) 0.1 MS Modified Vitamins
(1000X)* 1 ml pH 5.8 .sup.1All media contain basal salts (MS basal
salts) from Murashige and Skoog (1962) medium .sup.2MS Modified
medium (MS-BMS) *MS Modified (MS-BMS) Vitamins 1000X stock
Ingredient Amount/Liter Nicotinic Acid 650 mg Pyridoxine HCl 125 mg
Thiamine HCl 125 mg Ca Pantothenate 125 mg
[0125]
10TABLE 10 Protocol for Agrobacterium-Mediated Transformation of
Rice with nptII using G418 Selection 0d: Co-cultured on CC-1 1d:
End of co-culture and transferred to MS delay with 500 mg/L
Carbenicillin and 20 .mu.M AgNO3 4d: Removed coleoptile and
cultured on the same plate 7d: End of delay and transferred to
NPT-1, without sub-culture 15d: End of 1.sup.st selection.
Sub-cultured into small pieces and transferred to NPT-2
(pre-regeneration medium). Incubated in the dark 29d: Transferred
all callus pieces (without sub-culture) to NPT-3 (regeneration
medium). Incubated in the light at 23.degree. C. Petri- dishes were
placed in a clear storage container. Lighting conditions: 75-132
.mu.Mol photons m.sup.-2 .multidot. S.sup.-2 43d: Transferred all
green and regenerating pieces to NPT-4 (Plantcon) without excessive
sub-culture, Incubate in the light (same conditions as described
above) 60d Transferred to soil
[0126]
11TABLE 11 Protocol for Agrobacterium-Mediated Transformation of
Rice with CP4 Gene using Glyphosate Selection 0d: Co-cultured on
CC-1 1d: At the end of co-culture ransferred to MS delay with 500
mg/L carbenicillin and 20 .mu.M AgNO3. 4d: Removed coleoptile and
cultured on the same plate 7d: End of delay transferred to Gly-1,
without sub-culture. 15d: End of 1st selection. Transferred to
Gly-2 without sub- culture. Incubated in the dark. 22d: End of 2nd
selection. Sub-cultured into small pieces (.about.1 mm pieces) and
transferred to Gly-3. Incubated in the dark. 37d: Transferred all
callus pieces, (without sub-culture) to Gly-4 (regeneration
medium). Incubated in the light at 23.degree. C. Placed
petri-dishes directly in clear container. 52d: Transferred all
green and regenerating pieces Gly-5 (Plantcon) with excessive
sub-culture, (growth medium/Plantcon). Incubated in light. (75-132
.mu.Mol photons m.sup.-2 .multidot. S.sup.-2
[0127]
12TABLE 12 Supplemental Components in Basic Media used for Rice
Transformation Using CP4 Gene Components CC1 Delay Gly1 Gly2 Gly3
Gly4 Gly5 2,4-D (mg/L) 2.0 2.0 2.0 2.0 0.2 -- -- Picloram
(mg/L).sup.2 2.2 2.2 2.2 2.2 -- -- -- BAP (mg/L).sup.2 -- -- -- --
-- -- 2.0 -- Kinetin (mg/L).sup.2 -- -- -- -- -- -- 1.0 -- NAA
(mg/L).sup.2 -- -- -- -- -- -- 1.0 -- Glucose (g/L) 10 -- -- -- --
-- -- -- Sucrose (g/L) 20 20 20 20 20 60 60 Glutamine (g/L) -- 0.5
0.5 0.5 -- -- -- Magnesium Chloride -- 0.75 0.75 0.75 -- -- --
(g/L) Casein Hydrolysate -- 0.1 0.1 0.1 -- -- -- (g/L) L-Proline
115 -- -- -- -- -- -- -- (mg/L) Myo-Inositol (g/L) -- -- -- -- --
0.1 0.1 Thiamine HCl 0.5 1.0 1.0 1.0 -- -- -- (mg/L) ABA (mM) -- --
-- -- -- 0.2 -- -- Gelling agent (g/L) 5.5 (A) 2 (P) 2 (P) 2.0 (P)
2.5 (P) 2.5 (P) 2.5 (P) AgNO3 (M)* 20* -- 20* -- -- -- -- --
Carbenicillin -- 500 250 250 250 250 100 100 (mg/L) Glyphosate (mM)
-- -- 2.0 0.5 0.1 -- 0.05 mM pH 5.4 5.8 5.8 5.8 5.8 4.0 5.8 5.8
.sup.1All media contain basal salts (MS basal salts) and vitamins
(MS vitamins) from Murashige and Skoog (1962) medium.
.sup.2Filter-sterilized and were added to the medium after
autoclaving. .sup.3Low-EEO Agarose (A) or Phytagel .TM. (P).
*Amount AgNO3 added unless otherwise indicated in specific
examples.
[0128]
13TABLE 13 Protocol for Agrobacterium-Mediated Transformation of
Wheat with nptII using G418 Selection 0d: Co-cultured on 1/2 MSCC
1d: End of co-culture and transferred to W1 delay with 500 mg/L
carbenicillin and 20 .mu.M AgNO3 4d: Removed coleoptile and
cultured on the same plate 7d: End of delay and transferred to W2
without sub-culture 15d: End of 1.sup.st selection. Sub-cultured
into small pieces and transferred to W3 (pre-regeneration medium).
Incubated in the dark 29d: Transferred all regenerating callus
pieces (sub-culture) to W3 (pre-regeneration medium). Incubated in
light at 23.degree. C. (75- 132 .mu.Mol photons m.sup.-2 .multidot.
S.sup.-2). Placed plates directly in clear container. 43d:
Transferred all green and regenerating pieces to W4 (Plantcon)
without excessive sub-culture. Incubated in the light. 60d: Further
transferred all green and regenerating pieces to W4 (Plantcon)
without excessive sub-culture. Incubated in the light. 75d
Transferred plantlets to soil
[0129]
14TABLE 14 Supplemental Components in Basic Media used for Rice
Transformation Using nptII Components CC1 Delay NPT1 NPT2 NPT3 NPT4
2,4-D (mg/L) 2.0 2.0 2.0 0.2 -- -- Picloram (mg/L) 2.2 2.2 2.2 --
-- -- BAP (mg/L) -- -- -- -- 2.0 -- Kinetin (mg/L) -- -- -- -- 1.0
-- NAA (mg/L) -- -- -- -- 1.0 -- Glucose (g/L) 10 -- -- -- -- --
Sucrose (g/L) 20 20 20 20 60 60 Glutamine (g/L) -- 0.5 0.5 -- -- --
Magnesium Chloride -- 0.75 0.75 -- -- -- (g/L) Casein Hydrolysate
-- 0.1 0.1 -- -- -- (g/L) L-Proline 115 -- -- -- -- -- (mg/L)
Myo-Inositol (g/L) -- -- -- -- 0.1 0.1 Thiamine HCl 0.5 1.0 1.0 --
-- -- (mg/L) ABA (mM) -- -- -- 0.2 -- -- Gelling agent (g/L) 5.5
(A) 2 (P) 2 (P) 2.5 (P) 2.5 (P) 2.5 (P) AgNO3 (M) 20 20 -- -- -- --
Carbenicillin -- 500 250 250 100 100 (mg/L) G418 (mg/L) -- -- 40 40
40 40 pH 5.4 5.8 5.8 4.0 .sup.1All media contained basal salts (MS
basal salts) and vitamins (MS vitamins) from Murashige and Skoog
(1962) medium. .sup.2Filter-sterilized and were added to the medium
after autoclaving. .sup.3Low-EEO Agarose (A) or Phytagel .TM.
(P).
[0130]
15TABLE 15 Supplemental Components in Basic Media used for Wheat
Transformation Components 1/2MS CC W1 W2 W3 W4 2,4-D (mg/L) 3.0 0.5
0.5 0.2 -- Picloram (mg/L) -- 2.2 2.2 -- -- Maltose (g/L) -- 40 40
40 40 Glucose (g/L) 10 -- -- -- -- Sucrose (g/L) 20 -- -- -- --
Glutamine (g/L) -- 0.5 0.5 -- -- Magnesium Chloride -- 0.75 0.75 --
-- (g/L) Cascin Hydrolysate -- 0.1 0.1 -- -- (g/L) MES (g/L).sup.2
-- 1.95 1.95 1.95 1.95 Ascorbic Acid -- 100 100 100 100
(mg/L).sup.2 L-Proline 115 -- -- -- -- (mg/L) Thiamine HCl 0.5 --
-- -- -- (mg/L) Gelling agent 5.5 (A) 2 (P) 2 (P) 2 (G) 2 (G)
(g/L).sup.3 AgNO3 (.mu.M) 20 -- -- -- Carbenicillin -- 500 500 500
500 (mg/L) G418 (mg/L) -- -- 25 25 25 pH 5.4 5.8 5.8 5.8 5.8
.sup.1All media contained basal salts (MS basal salts) and vitamins
(MS vitamins) from Murashige and Skoog (1962) medium.
.sup.2Filter-sterilized and were added to the medium after
autoclaving. .sup.3Low-EEO Agarose (A) or Phytagel .TM. (P) or
Gelrite (G).
Example 7
[0131] Efficiency of T-DNA Delivery
[0132] The number of transgenic events in each study was determined
after the plants were assayed unless indicated otherwise. The
transformation efficiency (number of events/number of explants e.g.
immature embryos, varied from study to study and among different
treatment conditions and among different genotypes.
[0133] The efficiency of T-DNA delivery to different cell types are
described in more detail in the specific examples.
Example 8
[0134] Transgenic Plant Analyses
[0135] The plants were grown in a greenhouse under appropriate
growth conditions as described above. The majority of plants were
fully fertile. Each plant was examined by one or more of the
following methods:
[0136] a) The GUS histochemical assay (Jefferson, 1987) using
different parts of the plants.
[0137] b) Biological assay (leaf bleach assay). Leaf samples (a
leaf punch) from approximately 2-week-old seedlings were placed in
wells of 24-well cell culture clusters (Costar Corporation,
Cambridge, Mass.). Each well was filled with 0.5 ml aqueous
solution composed of 300 mg/L paromomycin (Sigma) and 100 mg/L
Benlate (a fungicide made by Du Pont), 100 mg/IL Benlate alone was
used as a control. Three leaf samples from the same leaf of each
plant were placed in two wells containing paromomycin plus Benlate
and one well containing Benlate alone, respectively. Leaf samples
from non-transformed plants were used as negative controls. The
samples were vacuum-infiltrated in a dessicator using an in-house
vacuum system for 5 min and then the clusters were sealed tightly
with parafilm before being placed under light (140 .mu.Mol m-2s-1).
The results were determined after 60 hours. The leaf samples that
were highly resistant to paromomycin remained green in most area
except the cut edges (<1 mm wide), which indicated that the
plants had the functional nptII gene. The leaf samples from the
plants without the gene or with the non-functional gene were
bleached out completely by paromomycin as the negative controls, or
had only small patches of green area.
[0138] c) Southern hybridization analysis (Southern, 1975). Genomic
DNA was isolated from leaf tissue of the plants following the
method of Shure et al. (1983). Ten to fifteen milligrams of genomic
DNA was digested with the appropriate restriction endonuclease and
fractionated on a 0.8% agarose gel. The DNA was transferred to
Hybond N membranes (Amersham, Arlington Heights, Ill.) according to
standard procedures (Sambrook et al., 1989). The probe for corn
plants transformed with pMON18365 (FIG. 2) and pMON25457 (FIG. 3)
was prepared by gel purifying a .about.1.5 kb fragment containing
35S-hsp fragment. Genomic DNA of corn lines was digested with
EcoRI. DNA from rice lines transformed with pMON32902 was digested
with XhoI and probed with a gel purified .about.1.6 kb fragment
from pMON25492 (FIG. 4) containing the CP4 gene. The probe was
labeled with 32P dCTP using a random primer labeling kit (Prime-It
II, Strategene, La Jolla, Calif.), to a specific activity of
2.6.times.109 cpm/mg. The membrane was hybridized for 14 hours at
42.degree. C. in a solution containing 50% formamide, 5.times.SSC,
5.times. Denhardt's, 0.5% SDS and 100 .mu.g/ml tRNA. The condition
of the final wash was 0.1% SSC and 0.1% SDS at 60.degree. C. for 15
minutes.
Example 9
[0139] Effect of the Addition of Growth Inhibiting Agents During
the Growth of Agrobacterium Cells on Transformation of Plant
Cells
[0140] Explant Preparation
[0141] Two explants are used for this study:
[0142] 1) Young leaves of kalanchoe plants grown in the green house
and
[0143] 2) rapidly growing BMS suspension cells.
[0144] Preparation of Agrobacterium Cells
[0145] Agrobacterium cells used for transformation were pre-induced
as described in Table 2 For transformation of kalenchoe leaves
Agrobacterium cells were washed in the MS inoculation medium
without any additives (only with salts). For the transformation of
BMS suspension cells, the standard protocol as described in Table 8
was followed.
[0146] Inoculation and Co-Cultivation
[0147] Transformation of BMS suspension cells was performed
following the protocol described in Table 8. For transforming
kalanchoe leaves, a suspension of cells from Agrobacterium strain
A281 was applied after performing mechanical wounding as described
in White and Nester. The Agrobacterium strain, A136 harboring a
binary vector pMON25457 (FIG. 3) was used as a negative
control.
[0148] Efficiency of T-DNA Delivery
[0149] The efficiency of T-DNA delivery to BMS cells was measured
by transient GUS expression post co-cultivtion as well as by
staining GUS positive colonies appearing on a single piece of
filter 4 weeks after co-culture.
[0150] The efficiency of T-DNA delivery to kalanchoe leaves was
determined by evaluating gall formation 4 weeks post-inoculation
using 20 mls of Agrobacterium A281 suspension cells.
Example 10
[0151] Effects of Addition of Growth Inhibiting Agent during
Pre-Induction of Agrobacterium Cells
[0152] Agrobacterium cells EHA 101:pMON25457 (for transforming BMS
cells) and A281 (for transforming kalanchoe leaves) were
pre-induced as described above in the AB medium. During
pre-induction, AgNO.sub.3 was added at two different levels (20M
and 40M final concentration) to the pre-induction medium. The final
optical density prior to the induction was adjusted to A.sub.660
(OD 0.2). Agrobacterium cells were pre-induced for 15 hours.
Measurement of the optical density (measure of growth) was taken at
the end of pre-induction, just prior to transformation.
Agrobacterium cells pre-induced in the absence of AgNO.sub.3 were
used as a control. The effect of AgNO.sub.3 during the
pre-induction stage on the growth and T-DNA transfer is shown in
Table 16, Table 17 and Table 18. The presence of AgNO.sub.3 during
growth of Agrobacterium cells prior to the transformation inhibits
the growth and T-DNA transfer ability of Agrobacterium cells.
Plating Agrobacterium cells on semi-solid LB plates indicated that
a 15 hour culture period of Agrobacterium in the presence of
AgNO.sub.3 was lethal to the Agrobacterium cells. Accordingly, no
stable transformants were obtained when Agrobacterium cells were
treated with the growth inhibiting agent AgNO.sub.3. Controls
produced tumors (strain A281 on kalenchoe plant tissue) and GUS
positive calli (strain EHA1010:pMON25457 on BMS suspension
cells)
16TABLE 16 Effect of AgNO.sub.3 on Growth of Agrobacterium Cells
(Pre-induction) Treatment OD A.sub.660 after 15 hours growth
Results minus AgNO.sub.3 0.54 growth plus 20M AgNO.sub.3 0.23 no
growth plus 40M AgNO.sub.3 0.24 no growth
[0153]
17TABLE 17 Effect of Addition of AgNO.sub.3 During Growth of
Agrobacterium on T-DNA Transfer (Tumor Induction) to Kalanchoe
Cells Treatment tumor formation Results minus AgNO.sub.3 + T-DNA
transfer plus 20M AgNO.sub.3 - no transfer plus 40M AgNO.sub.3 - no
transfer
[0154]
18TABLE 18 Effect of Addition of AgNO.sub.3 During Growth of
Agrobacterium on T-DNA Transfer to BMS cells (Pre-Induction)
Average # of GUS positive Treatment colonies/filter paper Results
minus AgNO.sub.3 76 T-DNA transfer plus 20M AgNO.sub.3 0 no
transfer plus 40M AgNO.sub.3 0 no transfer
Example 11
[0155] Effect of Presence of Growth Inhibiting Agent During the
Co-Culture Period on Agrobacterium Cell Growth
[0156] Plant Materials
[0157] Various corn explants e.g. immature embryos isolated
approximately 10 days after pollination and immature embryo derived
callus, both cultured on D medium (Duncan et al., 1985); callus
derived from immature embryos (TypeII callus derived from Hi-II
genotype) and cultured on modified N6 medium (Armstrong et al.,
1991); BMS suspension cells as described previously were used in
this study.
[0158] Agrobacterium Strains and Plasmids
[0159] Disarmed Agrobacterium tumefaciens strain C58 (ABI)
harboring binary vector pMON18365 (FIG. 2) was used in this
Example. The Agrobacterium strain was pre-induced as described
previously.
[0160] Inoculation and Co-Cultivation
[0161] Three mls of pre-induced Agrobacterium suspension (A.sub.660
OD 1.0) was added to a 6-well tissue culture plate. After adding
the explants, the plant tissues and Agrobacterium suspension cells
were subjected to vacuum infiltration for three hours. After the
three hour vacuum infiltration, the Agrobacterium suspension was
removed and the plant tissues were placed on semi-solid medium
containing 10M AgNO3 (final concentration). All the tissues were
incubated for three days in the dark.
[0162] Effect of AgNO3 During Co-Culture on Agrobacterium Cell
Growth
[0163] No growth of Agrobacterium cells surrounding explants was
observed on co-culture medium three days post-co-culture. All
explants were transferred to the medium without the growth
inhibiting agent and evaluation of Agrobacterium growth was
observed seven days post-transfer. Profuse growth of Agrobacterium
cells was noticed surrounding the explants. Thus, addition of the
growth inhibiting agent to the co-culture medium inhibited growth
of some bacterial cells, but did not kill of all Agrobacterium
cells under the conditions tested.
Example 12
[0164] Effect of the Presence of Growth Inhibiting Agent During
Co-Culture Period on the Recovery of Agrobacterium
[0165] In an another example, immature embryos were isolated as
described in Example 3. Disarmed Agrobacterium tumefaciens strain
EHA 101 harboring binary vector pMON25457 (FIG. 3) was used. The
Agrobacterium strain was pre-induced as described in Example 2.
Three mls of pre-induced Agrobacterium suspension (A.sub.660 OD
4.0) was added to a 6-well tissue culture plate as described. After
adding the explants to the Agrobacterium suspension cells, the
inoculation was performed for 15 minutes. The Agrobacterium
suspension was removed and the embryos were placed on semi-solid
medium with or without AgNO3 (20 M final concentration). All the
tissues were incubated for three days in the dark. The amount of
Agrobacterium present was estimated at the beginning by randomly
sampling immature embryos immediately after inoculation and again
at the end of the co-culture period to determine the number of
attached Agrobacterium cells per immature embryo explant. The
results are presented in Table 19. The results demonstrate that
inclusion of AgNO.sub.3 during co-culture significantly inhibited
the growth of Agrobacterium.
19TABLE 19 Presence of Growth Inhibiting Agent during Co-Culture
Period Reduces Agrobacterium Cell Proliferation During Co-Culture
Average # of Agrobacterium Treatment Colonies/Explant Results 0d 3
.times. 10.sup.5 CFU* 3d minus 20M AgNO.sub.3 2.0 .times. 10.sup.6
CFU 6.7 fold increase 3d plus 20M AgNO.sub.3 1.0 .times. 10.sup.4
CFU 30 fold reduction *CFU = colony forming units
Example 13
[0166] Pre-Induction of Agrobacterium Optimizes T-DNA Delivery When
Co-Cultured in the Presence of Growth Inhibiting Agents
[0167] Immature embryos of corn genotype H99.times.A188 and
Agrobacterium strain ABI harboring binary vector pMON 18365 (FIG.
2) were used. The Agrobacterium strain was pre-induced as described
previously and three mls of pre-induced Agrobacterium suspension
(A.sub.660 OD 1.0, 2.0, 3.0 and 4.0) was used. After adding the
explants the Agrobacterium suspension cells, the inoculation was
performed for 15 minutes. The Agrobacterium suspension was removed
and embryos were placed on {fraction (1/2)} MS corn co-culture
medium supplemented with 10M AgNO3 (final concentration). The
co-culture duration was for three days in the dark. The efficiency
of T-NA delivery was estimated by a transient GUS analysis three
days after co-culture by incubating embryos directly in GUS
staining buffer for 12-15 hours and counting the number of GUS foci
per immature embryo explant (Table 20). Increasing the
concentration of Agrobacterium cells had no effect on the frequency
of T-DNA transfer to corn tissues when Agrobacterium cells were
grown in LB medium. For the pre-induction stage treatment, T-DNA
transfer as measured by transient GUS expression increased as
Agrobacterium concentration increased from an OD.sub.660 of 1.0 to
4.0.
20TABLE 20 Pre-induction of Agrobacterium Optimizes T-DNA delivery
When Co-Cultured in the Presence of AgNO.sub.3 Treatment Average
number of OD.sub.660 Induction state GUS foci/explant 1.0
Pre-induced 7 2.0 Pre-induced 28 3.0 Pre-induced 39 4.0 Pre-induced
66 2.0 Not pre-induced (grown in LB) 2 4.0 Not pre-induced (grown
in LB) <1
Example 14
[0168] Effect of Presence of Growth Inhibiting Agent During
Co-Culture on T-DNA Transfer and Plant Cell Growth
[0169] Immature embryos of genotype H99.times.A188 were isolated as
described above. The disarmed Agrobacterium strain ABI harboring
binary vector pMON18365 (FIG. 2) was used. The Agrobacterium strain
was pre-induced as described above and three mls of pre-induced
Agrobacterium suspension (A.sub.660 OD 4.0) was added to a 6-well
tissue culture plate as described earlier. The inoculation was
performed for three hours under vacuum. The Agrobacterium
suspension was removed and embryos were placed on respective
semi-solid medium containing various concentrations of AgNO3 (0,
10, 20, 40, 60 M AgNO.sub.3 final concentration). All the tissues
were incubated for three days in the dark. The efficiency of T-DNA
delivery was estimated by a transient GUS analysis performed three
days after co-culture by counting the number of GUS foci per
immature embryo explant (Table 21). Efficiency of culture response
was determined by transferring the embryos to a delay medium (D
medium, supplemented with 500 mg/L Carbenicillin) and taking
observation 2 weeks post transfer. The presence of 10M AgNO.sub.3
during co-culture had a positive effect on both the frequency of
T-DNA transfer as measured by the average number of GUS foci and
tissue survival. Increasing the levels of AgNO.sub.3 to 20M
decreased the amount of T-DNA transfer but increased the frequency
of the embryos responding to the culture. Increasing the level of
AgNO.sub.3 to 60M was found to be inhibitory to T-DNA transfer but
the higher level did not appreciably increase the culture response.
The results demonstrate that the concentration of an growth
inhibiting agent such as AgNO3 can be titrated to obtain the
desired efficiency of T-DNA transfer.
21TABLE 21 Manipulation of T-DNA Transfer with Addition of Growth
Inhibiting Agent During Co-Culture % of Average number of immature
embryos Treatment GUS foci/explant responding to culture minus
AgNO.sub.3 46 29 plus 10M AgNO.sub.3 63 42 plus 20M AgNO.sub.3 26
64 plus 40M AgNO.sub.3 28 62 plus 60M AgNO.sub.3 12 60
Example 15
[0170] Reduction of Agrobacterium Density During Co-Culture Using a
Growth Inhibiting Agent Increases the Frequency of Transformation
of Corn, Rice and Wheat
[0171] Explant Preparation
[0172] The explants used in this study were immature embryos and
were prepared as described previously.
[0173] Agrobacterium Transformation and Selection:
[0174] The following transformation protocols included the
following parameters: use of immature embryos that were
pre-cultured for less than 24 h; a bacterial inoculation density
>2.0 at OD.sub.660, a co-culture duration of from one to three
days, the use of a higher concentration of auxin or different type
of auxin/combination of growth regulators than that required during
normal tissue culture, a delay period 3-7d following co-culture
(with out selection pressure), no sub-culture of the original
explant, a step-wise increase or decrease, depending on the crop
and selection scheme and a transformation duration between 9-12
weeks.
22TABLE 22 Transformation Efficiency Increases with the Addition of
Growth Inhibiting Agent During Co-Culture in Corn, Rice, and Wheat
Treatment Transformation (%) corn: OD.sub.660 2.0 plus 20M
AgNO.sub.3 18 (8/45) OD.sub.660 2.0 minus 20M AgNO.sub.3 4 (2/51)
OD.sub.660 4.0 plus 20M AgNO.sub.3 8 (4/52) OD.sub.660 4.0 minus
20M AgNO.sub.3 2 (1/48) (co-culture duration was three days) rice:
OD.sub.660 2.0 plus 20 mMAgNO.sub.3 23 (5/21) OD.sub.660 2.0 minus
20 mMAgNO.sub.3 4 (4/111) (co-culture duration was one day) wheat:
OD.sub.660 4.0 plus 20 mMAgNO.sub.3 4 (1/25) OD.sub.660 4.0 minus
20 mMAgNO.sub.3 0 (0/22) (co-culture duration was three days)
Example 16
[0175] Effects of Addition of Growth Inhibiting Agents to
Inoculation Media
[0176] Corn genotype LH198 X Hi-II was used. Corn immature embryos
were isolated as described previously. Approximately 30 immature
embryos were inoculated for each treatment with Agrobacterium
strain ABI harboring plasmid pMON18365 for five minutes and placed
on co-culture media for two to three days. There were 4 replicates
per treatment. The four treatments included:
[0177] Treatment 1: absence of growth inhibiting agent (in both
inoculation and co-culture media)
[0178] Treatment 2: absence of agent in inoculation media; presence
of agent (20 .mu.M silver nitrate) in co-culture media.
[0179] Treatment 3: presence of agent (20 .mu.M silver nitrate) in
inoculation media; absent in co-culture media
[0180] Treatment 4: presence of agent (20 .mu.M silver nitrate) in
both inoculation and co-culture media
[0181] Within each treatment, five immature embryos were used for
transient analysis. This was repeated across all reps within each
treatment. The number of GUS spots was determined on both the
scutellar surface (scutellar side up) as shown in Table 23 and the
number of GUS spots was determined on the axis side of the embryos
as shown in Table 24. The results demonstrated that the presence of
the Agrobacterium inhibitory growth agent in the inoculation medium
and co-culture medium decreased the number of GUS spots compared
with the presence of the agent in either inoculation or the
co-culture medium or without the inhibitory agent. Thus, presence
of an Agrobacterium growth inhibiting agent in the inoculation
stage and during the co-culture stage can be used to decrease T-DNA
transfer and copy number. The T-DNA transfer process was also
influenced by the orientation of the tissue with respect to the
location of the inhibitory agent, as demonstrated by the decrease
in the number of GUS spots on the axis side of the tissue which is
the bottom surface of the tissue (closest to the growth inhibiting
agent on the co-culture plate) (Table 24)
23TABLE 23 Effects of Presence or Absence of Growth Inhibiting
Agent in Inoculation Media (MS-PL) and/or Co-culture Media (1/2 MS
CC) on Transient GUS Expression Average # GUS spots/embryo
Treatment (scutellar surface) Duncan Grouping* 1 (none) 80.4 A 2
(inoculation) 82.5 A 3 (co-culture) 81.8 A 4 (both) 58.9 A *Means
with same letter indicate no significant difference according to
Duncan's New Multiple Range Test at a 5% probability level.
[0182]
24TABLE 24 Effects of Presence or Absence of Growth Inhibiting
Agent in Inoculation Media (MS-PL) and/or Co-culture Media (1/2 MS
CC) on Transient GUS Expression Average # GUS Treatment
spots/embryo (axis) Duncan Grouping* 1 (none) 39.1 A 2
(inoculation) 40.8 A 3 (co-culture) 3.8 B 4 (both) 8.4 B *Means
with same letter indicate no significant difference according to
Duncan's New Multiple Range Test at a 5% probability level.
Example 17
[0183] Addition of Growth Inhibiting Agent During Inoculation
Improves Transformation Efficiency in Corn.
[0184] Immature embryos of genotype (H99.times.Pa 91)A188 were
isolated as described. The Agrobacterium strain EHA 101 harboring
binary vector pMON25457 (FIG. 3) was used. The Agrobacterium strain
was pre-induced as described and three mls of pre-induced
Agrobacterium suspension (A.sub.660 OD 0.5) supplemented with 20M
AgNO3 was added to a 6-well tissue culture plate as described
above. Inoculation was performed for 15 minutes. The Agrobacterium
suspension was removed and embryos were placed on semi-solid
co-culture medium containing 20 M AgNO.sub.3 (final concentration).
All the tissues were incubated for three days in the dark. The
transformation protocol followed as described in Example 6. Control
embryos were cultured in the absence of AgNO.sub.3 in all steps of
the transformation process. Transformation efficiency was
calculated based on the number of embryos producing paromomycin
resistant calli. The results demonstrate that the addition of a
growth inhibition agent such as AgNO3 during inoculation increases
the transformation efficiency.
25TABLE 25 Inclusion of Growth Inhibiting Agent AgNO.sub.3 During
Inoculation Improves Transformation Efficiency in Corn* Treatment
condition % Transformation* No AgNO.sub.3 1.5 (1/65) 5d 20 M
AgNO.sub.3.sup.*1 7.8 (4/51) 8d 20 MAgNO.sub.3.sup.*2 9.0 (5/55)
*data in the parenthesis indicates total number of embryos
producing Paromomycin positive events / total number of embryos
inoculated. *.sup.1AgNO.sub.3 was not present during 3 day
co-culture period but was present during inoculation and 5 day
delay period following co-culture. .sup.*2AgNo.sub.3 was present
during inoculation, 3 day co-culture and 5 day delay period.
Example 18
[0185] Effects of Other Chemicals on Growth of Agrobacterium
[0186] Each of the chemicals listed in Table 26 was resuspended in
MS-BMS media and added (final concentration 50 .mu.M) to a 50 ml
overnight culture of Agrobacterium (strain ABI). Twenty-four hours
after inoculation the effect of the chemicals on the growth of
Agrobacterium was recorded. A known bacteriocidal compound,
Carbenicillin at a final concentration of 50 mg/L and AgNO3 (20
.mu.M) were used as controls. "No Growth" indicates there was not
an increase in cell density indicating bacteriocidal or
bacteriostatic property of the chemical, "Slow Growth" indicates
that a slight increase in cell density was noticed and a higher
level may be lethal. "Growth" indicates no effect on bacterial
growth at the concentration used relative to growth in control
medium, and a higher concentration may be needed to elicit an
effect on growth.
26TABLE 26 Bacteriocidal or bacteriostatic properties of different
chemicals on Agrobacterium Chemical Effect Aluminum Chloride Growth
Cadmium Chloride Slow Growth Chromium (II) Chloride Growth Lead
Nitrate Growth Manganese Chloride Slow Growth Nickel Chloride
Growth Potassium Chromate No Growth Silver Nitrate No Growth Sodium
Molybdate Growth Sodium Tungstate Growth Zinc Chloride Growth
Carbenicillin No Growth
Example 19
[0187] Improvements in Transformation of Corn by Reduction of
Agrobacterium Density During Co-Culture with Different Growth
Inhibiting Agents
[0188] Immature embryos of genotype (H99.times.Pa 91)A188 were
isolated as described. The Agrobacterium strain EHA 101 harboring
binary vector pMON25457 (FIG. 3) was used. The Agrobacterium strain
was pre-induced as described. The inoculation was performed using a
concentration of A.sub.660 OD 4.0 for 15 minutes as described. Post
inoculation, Agrobacterium suspension was removed and embryos were
placed on different semi-solid co-culture media supplemented with
various bacteriocidal compounds. All the tissues were incubated for
three days in the dark. The transformation protocol followed was as
described previously (corn IE transformation) except that the
1.sup.st selection with 50 mg/L Paromomycin was replaced with 25
mg/L Paromomycin for 2 weeks and 50 mg/L Paromomycin for 2
additional weeks. Transformation efficiency was calculated based on
the number of embryos producing nptII positive plants as determined
by a leaf bleach assay as described earlier. Three weeks after the
transformation, the quality and growth characteristics of
IE-derived callus co-cultured in the presence of different growth
inhibiting agents. The culture response on different co-culture
media containing different agents was as follows: 50 .mu.M AgNO3
produced embryogenically the most competent callus>20 .mu.M
AgNO3>Carbenicillin>without additives>K2CrO4 produced
embryogenically less competent callus. The results demonstrate that
a higher frequency of transformation can be obtained when an growth
inhibiting agent such as silver nitrate is added during the
co-culture period. A decreased level of transformation (reduced
frequency of T-DNA transfer) was obtained when the concentration of
AgNO3 was increased from 20 .mu.M to 50 .mu.M, although a higher
culture response was achieved. Addition of K2CrO4 was detrimental,
presumably due to extreme negative effects of this chemical on
plant cell health in addition to the effects of the chemical on
Agrobacterium. The data demonstrated that the increase in
transformation effciency was related to inhibiting the
Agrobacterium growth during the co-culture rather than the
improvements in the culture response.
27TABLE 27 Transformation Efficiency Improvements of Corn by Using
Different Growth Inhibiting Agents During Co-Culture Treatment %
Transformation* 50 M AgNO.sub.3 6.3 (6/95) 20 MAgNO.sub.3 16 (9/56)
50 M K2CrO4 0 (0/104) Carbenicillin (50 mg/L) 14.3 (5/35) no
chemicals 2.6 (2/77) *data in the parentheses indicate total number
of embryos producing paromomycin positive events / total number of
embryos inoculated.
Example 20
[0189] Novel Explants for Transforming Cereals with Agrobacterium:
Improvements in Transformation of Corn by Using Hybrid Embryos
Containing Three Genotypes
[0190] Hybrid corn embryos were used to test the effect of an
growth inhibiting agent for improving the transformation process.
The data presented in the Table 28 demonstrated that the use of a
faster dividing cell line can increase the frequency of
transformation. Furthermore, faster cell division may allow the
selection/elimination of transgenic events containing complex or
multiple copies of inserts.
[0191] Immature embryos of different corn genotypes e.g. H99,
H99.times.A188 and (H99.times.Pa 91)A188 were isolated as
described. The Agrobacterium strain EHA 101 harboring binary vector
pMON25457 (FIG. 3) or ABI harboring binary vector pMON18365 (FIG.
2) were used. Agrobacterium strain was pre-induced as described the
inoculation was performed using a concentration of A.sub.660 OD 4.0
for 15 minutes as described. Post-inoculation, the Agrobacterium
suspension was removed and embryos were placed on semi-solid
co-culture medium supplemented with 20M AgNO3. All the tissues were
incubated for three days in the dark. The transformation protocol
followed was as described previously except that with the genotype
H99 the 1.sup.st selection with 50 mg/L paromomycin was replaced
with 25 mg/L paromomycin for two weeks and 50 mg/L paromomycin for
two additional weeks. Transformation efficiency was calculated
based on the number of embryos producing nptII positive plants as
determined by a leaf bleach assay as described earlier. It is
evident from the data presented in the Table 28 that the use of a
faster dividing cell line containing 3 genotypes produced a higher
frequency of transformation
28TABLE 28 Improvements in Transformation of Corn by Using Hybrid
Embryos Explants Containing Three Genotypes and a Growth Inhibiting
Agent Genotype % Transformation* H99 2.4 (4/164).sup.2 H99 1.2
(8/683).sup.2 H99 1.0 (8/745).sup.2 H99XA188 1.7 (2/114).sup.1
(H99XPa91)A188 4.9 (25/508).sup.1 (H99XPa91)A188 12.2
(50/409).sup.2 *data in the parenthesis indicate the total number
of embryos producing paromomycin positive events / total number of
embryos inoculated. .sup.1ABI:pMON18365 .sup.2EHA101:pMON25457
Example 23
[0192] Production of Transgenic Events with Lower Copy Number
Inserts Using Bacteriocidal Compounds During the Co-Culture
Medium.
[0193] Immature embryos of corn and rice were transformed with the
Agrobacterium strain ABI 101 harboring the binary vector pMON18365
(FIG. 2) and EHA 101 harboring the binary vector pMON32092 (FIG. 5)
using methods containing an growth inhibiting agent during the
co-culture as described above. The analysis of copy number was
performed using Southern hybridization as previously described. The
data presented in the Table 29 demonstrated that the use of a
growth inhibiting agent resulted in the production plants with 1-2
copy number of inserts at a very high frequency compared to what
has been achieved with other transformation system published to
date (Hiei et al., 1994 and Isida et al., 1996).
29TABLE 29 Reduction of Agrobacterium Density During Co-Culture
Increases Frequency of Stable Transformation of Corn and Rice Copy
number Crop Vector % 1 insert % 2 inserts corn ABI:pMON18365 83
(15/18) 17 (3/18) rice EHA101:pMON32092 42 (21/50) 42 (21/50)
Example 22
[0194] Production of Transgenic Events with Higher Co-Expression of
the Reporter Gene
[0195] Immature embryos of corn and rice were transformed with the
Agrobacterium strains ABI 101 harboring the binary vector pMON18365
(FIG. 2) and EHA 101 harboring the binary vector pMON25457 (FIG. 3)
using methods including an growth inhibiting agent during the
co-culture as described. The efficiency of co-transformation was
determined by determining the number of nptII positive plants
expressing GUS using histochemical staining as described. The data
presented in the Table 30 demonstrated that the use of an growth
inhibiting agent resulted in the production of plants with a high
co-expression frequency.
30TABLE 30 Production of Transgenic Events with Higher
Co-Expression of the Reporter Gene Crop Vector % Co-expression corn
EHA101:pMON25457 98 (98/107) rice EHA101:pMON25457 88 (30/34)
Example 23
[0196] Production of Transgenic Events with Higher Co-Expression of
the Reporter Gene
[0197] Immature embryos of corn of two different genotypes were
transformed with the Agrobacterium strain EHA 101 harboring the
binary vector pMON25457 (FIG. 3) using methods containing an growth
inhibiting agent during the co-culture as described above. The
segregation analysis were performed germinating immature embryos of
corn 12-14 days post controlled pollination (back crossing) on MSOD
medium containing 100 mg/L Paromomycin. The data presented in the
Table 31 demonstrated that the use of an growth inhibiting agent
resulted in the production of plants with higher events with the
presence of transgene at a single locus. Evidence was presented
earlier that the majority of this locus contain lower copy inserts
(>50% single copy for rice and >87 for corn). Furthermore, it
is also evident from the results that combination of 3 or more
genotypes results in a higher number of plants with single
segregating locus than H99, supporting our earlier results that
faster cell division allowed the selection/elimination of
transgenic events containing complex or multiple copies of
inserts.
31TABLE 31 Production of transgenic events with simple segregation
pattern in corn segregating locus Genotype vector % single* %
>single* (H99xPa91)A188 EHA101:pMON25457 90 (69/77) 10 (8/77)
H99 EHA101:pMON25457 80 (16/20) 20 (4/20)
Example 24
[0198] Higher Concentration of Auxin(s) with Addition of Growth
Inhibiting Agent Improves the Transformation Efficiency in Rice
[0199] Immature embryos of rice were transformed with the
Agrobacterium strain EHA 101 harboring the binary vector pMON25457
(FIG. 3) using methods including an growth inhibitory agent, 20 M
AgNO3 during the co-culture as described. The transformation
efficiency was determined on nptII positive events/total number of
embryos inoculated as previously described. The results
demonstrated that the combination of auxins or using 2 mg/L of
2,4-D with the addition of picloram during co-culture improves
transformation efficiency. Furthermore, the corn transformation
protocol described earlier used 3 mg/L of 2,4-D in the co-culture
medium, a level that is often too high for embryogenic callus
induction as well as regular maintenance of embryogenic callus of
corn.
32TABLE 32 Higher Concentration of Auxin(s) Improves the
Transformation Efficiency in IEs of Rice* Plasmid Vector Co-culture
medium** % Transformation EHA101:pMON25457 CC1 21 (23/108)
(EHA101:pMON25457 CC2 12 (14/118) *EHA101:pMON25457 **media recipe
in Table 33
[0200]
33TABLE 33 Supplemental Components in Basic Media used During
Co-culture of Rice Immature Embryos (IEs)*.sup.1 Components CC1 CC2
2,4-D (mg/L) 2.0 2.0 Picloram (mg/L) 2.2 -- *All other components
of the media are similar to 1/2 MSCC. .sup.1All media contain basal
salts (MS Basal Salts) and vitamins (MS vitamins) from Murashige
and Skoog (1962) medium. .sup.2Filter-sterilized and were added to
the medium after autoclaving.
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* * * * *