U.S. patent application number 10/336980 was filed with the patent office on 2003-07-17 for methods for enhancing plant transformation frequencies.
This patent application is currently assigned to Pioneer Hi-Bred International, Inc.. Invention is credited to Bidney, Dennis L., Church, Laura A., Gordon-Kamm, William J., Hill, Patrea M., Hoerster, George J., Lowe, Keith S., Ross, Margit C..
Application Number | 20030135889 10/336980 |
Document ID | / |
Family ID | 24455841 |
Filed Date | 2003-07-17 |
United States Patent
Application |
20030135889 |
Kind Code |
A1 |
Ross, Margit C. ; et
al. |
July 17, 2003 |
Methods for enhancing plant transformation frequencies
Abstract
The invention provides improved transformation methods. In
particular the method provides increased transformation frequency,
especially in recalcitrant plants. The method comprises stably
transforming a target cell with at least one polynucleotide of
interest. The target cell has been previously transformed to
stimulate growth of the cell and has gone through at least one cell
division.
Inventors: |
Ross, Margit C.; (Johnston,
IA) ; Church, Laura A.; (Des Moines, IA) ;
Hill, Patrea M.; (Des Moines, IA) ; Gordon-Kamm,
William J.; (Urbandale, IA) ; Lowe, Keith S.;
(Johnston, IA) ; Hoerster, George J.; (Des Moines,
IA) ; Bidney, Dennis L.; (Urbandale, IA) |
Correspondence
Address: |
PIONEER HI-BRED INTERNATIONAL INC.
7100 N.W. 62ND AVENUE
P.O. BOX 1000
JOHNSTON
IA
50131
US
|
Assignee: |
Pioneer Hi-Bred International,
Inc.
|
Family ID: |
24455841 |
Appl. No.: |
10/336980 |
Filed: |
January 6, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10336980 |
Jan 6, 2003 |
|
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09613094 |
Jul 10, 2000 |
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6512165 |
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Current U.S.
Class: |
800/288 ;
435/468; 800/312; 800/320.1 |
Current CPC
Class: |
C12N 15/8205 20130101;
C12N 15/8207 20130101; C12N 15/8201 20130101 |
Class at
Publication: |
800/288 ;
435/468; 800/312; 800/320.1 |
International
Class: |
C12N 015/87; A01H
005/00 |
Claims
What is claimed is:
1. A method for transforming a target plant cell comprising stably
transforming the target cell with a growth stimulation
polynucleotide to produce a modified cell, growing the modified
cell through at least one cell division to produce a progeny cell
expressing the growth stimulation polynucleotide, and then
transforming the progeny cell with at least one polynucleotide of
interest, wherein the growth stimulation polynucleotide is an
anti-apoptosis polynucleotide other than baculovirus p35 or
baculovirus iap; a hormone polynucleotide; or a silencing construct
targeted against cell cycle repressors.
2. The method of claim 1 wherein transformation frequency is
increased compared to corresponding plant cells that do not contain
the growth stimulation polynucleotide.
3. The method of claim 1 wherein the progeny cell is from T0
transgenic cultures, regenerated plants or any subsequent progeny
cell expressing the growth stimulation polynucleotide.
4. The method of claim 3 wherein the progeny cell is from a T0
regenerated plant or a plant from any subsequent generation
expressing the growth stimulation polynucleotide.
5. The method of claim 1 wherein the growth stimulation
polynucleotide is an anti-apoptosis polynucleotide other than
baculovirus p35 or baculovirus iap.
6. The method of claim 1 wherein the growth stimulation
polynucleotide is a hormone polynucleotide.
7. The method of claim 1 wherein the growth stimulation
polynucleotide is a silencing construct targeted against cell cycle
repressors.
8. The method of claim 1 wherein the target cell is from a monocot
or a dicot plant.
9. The method of claim 8 wherein the target cell is from a
monocot.
10. The method of claim 9 wherein the target cell is from a maize
plant.
11. The method of claim 8 wherein the target cell is from a
dicot.
12. The method of claim 11 wherein the target cell is from a
soybean plant.
13. The method of claim 8 wherein the target cell is from corn,
soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice,
barley, potato, tomato, or millet.
14. A method of transformation comprising transforming a target
plant cell with at least one polynucleotide of interest, wherein
the target cell has been previously stably transformed to express a
growth stimulation polynucleotide and has gone through at least one
cell division, wherein the growth stimulation polynucleotide is an
anti-apoptosis polynucleotide other than baculovirus p35 or
baculovirus iap; a hormone polynucleotide; or a silencing construct
targeted against cell cycle repressors.
15. The method of claim 14 wherein the target cell is from T0
transgenic cultures, regenerated plants or any subsequent progeny
expressing the transgenic growth stimulation polynucleotide.
16. The method of claim 15 wherein the target cell is from a T0
regenerated plant or any subsequent progeny expressing the
transgenic growth stimulation polynucleotide.
17. The method of claim 14 wherein the growth stimulation
polynucleotide is an anti-apoptosis polynucleotide other than
baculovirus p35 or baculovirus iap.
18. The method of claim 14 wherein the growth stimulation
polynucleotide is a hormone polynucleotide.
19. The method of claim 14 wherein the growth stimulation
polynucleotide is a silencing construct targeted against cell cycle
repressors.
20. The method of claim 14 wherein the target cell is from a
monocot or a dicot plant.
21. The method of claim 20 wherein the target cell is from a
monocot.
22. The method of claim 21 wherein the target cell is from a maize
plant.
23. The method of claim 20 wherein the target cell is from a
dicot.
24. The method of claim 23 wherein the target cell is from a
soybean plant.
25. The method of claim 20 wherein the target cell is from corn,
soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice,
barley, potato, tomato, or millet.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of co-pending application
U.S. Ser. No. 09/613,094 filed Jul. 10, 2000.
TECHNICAL FIELD
[0002] The present invention relates generally to plant molecular
biology.
BACKGROUND OF THE INVENTION
[0003] Cell division plays a crucial role during all phases of
plant development. The continuation of organogenesis and growth
responses to a changing environment requires precise spatial,
temporal and developmental regulation of cell division activity in
meristems (and in cells with the capability to form new meristems
such as in lateral root formation). Such control of cell division
is also important in organs themselves (i.e. separate from
meristems per se), for example, in leaf expansion and secondary
growth.
[0004] A complex network controls cell proliferation in eukaryotes.
Various regulatory pathways communicate environmental constraints,
such as nutrient availability, mitogenic signals such as growth
factors or hormones, or developmental cues such as the transition
from vegetative to reproductive. Ultimately, these regulatory
pathways control the timing, frequency (rate), plane and position
of cell divisions.
[0005] Plants have unique developmental features that distinguish
them from other eukaryotes. Plant cells do not migrate, and thus
only cell division, expansion and programmed cell death determine
morphogenesis. Organs are formed throughout the entire life span of
the plant from specialized regions called meristems.
[0006] In addition, many differentiated cells have the potential to
both dedifferentiate and to reenter the cell cycle. The study of
plant cell cycle control genes is expected to contribute to the
understanding of these unique phenomena. O. Shaul et al.,
Regulation of Cell Division in Arabidopsis, Critical Reviews in
Plant Sciences, 15(2):97-112 (1996).
[0007] Current transformation technology provides an opportunity to
engineer plants with desired traits. Major advances in plant
transformation have occurred over the last few years. However, in
many major crop plants, serious genotype limitations still exist.
Transformation of some agronomically important crop plants
continues to be both difficult and time consuming.
[0008] For example, it is difficult to obtain a culture response
from some maize genotypes. Typically, a suitable culture response
has been obtained by optimizing medium components and/or explant
material and source. This has led to success in some genotypes.
While, transformation of model genotypes is efficient, the process
of introgressing transgenes into production inbreds is laborious,
expensive and time consuming. It would save considerable time and
money if genes could be more efficiently introduced into and
evaluated directly into inbreds.
[0009] There is evidence to suggest that cells must be dividing for
transformation to occur. It has also been observed that dividing
cells represent only a fraction of cells that transiently express a
transgene. Furthermore, the presence of damaged DNA in non-plant
systems (similar to DNA introduced by particle gun or other
physical means) has been well documented to rapidly induce cell
cycle arrest (W. Siede, Cell cycle arrest in response to DNA
damage: lessons from yeast, Mutation Res. 337(2:73-84). Methods for
increasing the number of dividing cells would therefore provide
valuable tools for increasing transformation efficiency.
[0010] Current methods for genetic engineering in maize require a
specific cell type as the recipient of new DNA. These cells are
found in relatively undifferentiated, rapidly growing meristems, in
callus, in suspension cultures, or on the scutellar surface of the
immature embryo (which gives rise to callus). Irrespective of the
delivery method currently used, DNA is introduced into literally
thousands of cells, yet transformants are recovered at frequencies
of 10.sup.-5 relative to transiently expressing cells.
[0011] Exacerbating this problem, the trauma that accompanies DNA
introduction directs recipient cells into cell cycle arrest and
accumulating evidence suggests that many of these cells are
directed into apoptosis or programmed cell death. (Reference Bowen
et al., Tucson International Mol. Biol. Meetings). Therefore it
would be desirable to provide improved methods capable of
increasing transformation efficiency in a number of cell types.
[0012] While advances have been made in the transformation of elite
inbreds of maize, it would be desirable to increase frequencies of
transformation. Present model systems, designed around fast growing
and highly embryogenic cultures, produce high frequencies of
transgenic events in the hybrid GS3 and in model maize inbreds.
Because of the high frequencies, these models, instead of the elite
inbred genotypes, are frequently the standard target germplasm for
product development.
SUMMARY OF THE INVENTION
[0013] The present invention provides a method for increasing
transformation frequencies, especially in recalcitrant plants or
explants. The method comprises transforming a target cell with at
least one polynucleotide of interest operably linked to a promoter.
The target cell has previously been stably modified to stimulate
growth of the cell and has gone through at least one cell
division.
DETAILED DESCRIPTION OF THE INVENTION
[0014] Definitions
[0015] As used herein "promoter" includes reference to a region of
DNA upstream from the start of transcription and involved in
recognition and binding of RNA polymerase and other proteins to
initiate transcription.
[0016] As used herein, "polypeptide" and "protein" are used
interchangeably and mean proteins, protein fragments, modified
proteins, amino acid sequences and synthetic amino acid sequences.
The polypeptide can be glycosylated or not.
[0017] As used herein, "polynucleotide" and "nucleic acid" are used
interchangeably. A polynucleotide can be full-length or a fragment
and includes polynucleotides that have been modified for stability.
Unless otherwise indicated, the term includes reference to a
specific sequence or its complement.
[0018] As used herein, "growth stimulation polynucleotide" means a
polynucleotide capable of influencing growth of a cell. The
polynucleotides fall into several categories, 1) cell cycle
stimulatory polynucleotides 2) developmental polynucleotides 3)
anti-apoptosis polynucleotides other than baculovirus p35 or
baculovirus iap 4) hormone polynucleotides or 5) silencing
constructs targeted against cell cycle repressors.
[0019] The following are provided as examples of each category and
are not considered a complete list of useful polynucleotides for
each category: 1) cell cycle stimulatory polynucleotides including
plant viral replicase genes such as RepA, Cyclins, E2F, prolifera,
cdc2 and cdc25; 2) developmental polynucleotides such as Lec1, Kn1
family, WUSCHEL, Zwille, and Aintegumenta (ANT); 3) anti-apoptosis
polynucleotides other than baculovirus p35 or baculovirus iap such
as CED9, Bcl2, Bcl-X(L), Bcl-W, A1, McL-1, Mac1, Boo,
Bax-inhibitors; 4) hormone polynucleotides such as IPT, TZS, Baby
Boom (BBM) and CKI-1; 5) Silencing constructs targeted against cell
cycle repressors, such as Rb, CKI, prohibitin, wee1, etc. or
stimulators of apoptosis such as APAF-1, bad, bax, CED-4,
caspase-3, etc. and repressors of plant developmental transitions
such as Pickle and WD polycomb genes including FIE and Medea. The
polynucleotides can be silenced by any known method such as
antisense, cosuppression, chimerplasty, or transposon
insertion.
[0020] As used herein, "growth stimulation vector" means a vector
capable of altering the expression of polynucleotides resulting in
growth stimulation.
[0021] As used herein, "plant" includes but is not limited to plant
cells, plant tissue, plant parts, and plant seeds.
[0022] As used herein "recalcitrant plant or explant" means a plant
or explant that is more difficult to transform than model systems.
In maize such a model system is GS3. Elite maize inbreds are
typically recalcitrant. In soybeans such model systems are Peking
or Jack.
[0023] As used herein "responsive target plant cell" is a plant
cell that exhibits increased transformation efficiency after
transformation with a growth stimulation vector compared to a
corresponding plant cell that has not been transformed with the
growth stimulation vector.
[0024] As used herein "Stable Transformation" refers to the
transfer of a nucleic acid fragment into a genome of a host
organism (this includes both nuclear and organelle genomes)
resulting in genetically stable inheritance. In addition to
traditional methods, stable transformation includes the alteration
of gene expression by any means including chimerplasty or
transposon insertion.
[0025] As used herein "Transient Transformation" refers to the
transfer of a nucleic acid fragment into the nucleus (or
DNA-containing organelle) of a host organism resulting in gene
expression without integration and stable inheritance.
[0026] As used herein "Modified cells" are cells that have been
transformed.
[0027] As used herein "Re-transformation" refers to the
transformation of a modified cell.
[0028] The present invention provides novel methods for
transformation and for increasing transformation frequencies. A
responsive target plant cell is stably transformed with at least
one growth stimulation vector to produce a modified target cell.
The modified target cell is grown under conditions to produce at
least one cell division to produce a progeny cell expressing the
growth stimulation vector and then the progeny cell is transformed
with one or more vectors containing a polynucleotide of interest
operably linked to a promoter.
[0029] In another aspect of the invention a method for increasing
transformation efficiency is provided comprising transforming a
target plant cell with one or more vectors containing at least one
polynucleotide of interest operably linked to a promoter, wherein
the target cell has been previously modified to stimulate growth of
the cell and the modified cell has gone through at least one cell
division.
[0030] The modified cells can be obtained from T0 transgenic
cultures, regenerated plants or progeny whether grown in vivo or in
vitro so long as they exhibit stimulated growth compared to a
corresponding cell that does not contain the modification. This
includes but is not limited to transformed callus, tissue culture,
regenerated T0 plants or plant parts such as immature embryos or
any subsequent progeny of T0 regenerated plants or plant parts.
[0031] Examples of polynucleotides for use in the growth
stimulation vector are discussed above and include Cyclin
polynucleotides such as Cyclin A, Cyclin B, Cyclin C, Cyclin D,
Cyclin E, Cyclin F, Cyclin G, and Cyclin H; E2F; Cdc25; RepA and
similar plant viral polynucleotides encoding replication-associated
proteins; apoptosis inhibitor genes other than baculovirus p35 or
baculovirus iap such as CED9, Bcl2, Bcl-X(L), Bcl-W, A1, McL-1,
Mac1, Bax inhibitors, and Boo; homeotic genes or genes that
stimulate in vitro growth, such as Lec1, WUS, FUS3, and members of
the Knotted family, such as Kn1, STM, OSH1, and SbH1; and cytokinin
genes such as IPT, TZS, CKI-1 or BBM. Polynucleotides also useful
for growth stimulation include those designed to diminish
expression or activity of repressors of the cell cycle such as Rb,
CKI, prohibitin, wee1 or of plant development such as PICKLE or FIE
(Fertilization-independent endosperm).
[0032] Polynucleotides that encode polypeptides involved in the
regulation of or can influence cell cycle division in plants can be
used in the growth stimulation vector. Examples include cyclins
(Doerner (1994) Plant Physiol. 106:823-827.), maize cdc2 (Colasanti
et al. (1991) PNAS 88:3377-3381), other cdc2 WO 99/53069, cdc25+
(Russell and Nurse (1986) Cell 45:145-153), the geminivirus RepA
gene (U.S. Ser. No. 09/257,131), plant E2F (Ramirez-Parra et al.
(1999) Nuc. Ac. Res. 27:3527-3533 and Sekine et al. (1999) FEBS
Lett. 460:117-122), the IPT gene of Agrobacterium tumefaciens
(Strabala et al. (1989) Mol. Gen. Genet. 216:388-394, Bonnard et
al. (1989) Mol Gen. Genet. 216:428438, DDBJ/EMBL/GenBank), TZS
(Beaty et al. (1986) Mol. Gen. Genet. 203:274-280, Akiyoshi et al.
(1985) Nucleic Acids Res. 13:2773-2788, Regier et al. (1989)
Nucleic Acids Res. 17:8885), CKI1 (Kakimoto (1996) Science
274:982-985), BBM (Boutilier et al., Plant Mol. Biol. Reporter
18(2):S11-4 (2000) and PSK.alpha. (Yang et al. (1999) PNAS
96:13560-13565), all of which are incorporated herein by
reference.
[0033] Using methods of the invention with selected proteins such
as Bcl-2 (Pedoraro et al. (1984) Proc. Nat. Ac. Sci.
81(22):7166-7170), CED9 (Hengartner et al., Cell 76:665-676, 1994),
Bcl-X(L) (Yang et al., Immunity 7:629-639, 1997), Bcl-W (Hamner et
al., Neuroscience 91:673-684, 1999), A1 (Craxton et al., Cell
Immunology 200:56-62, 2000), McL-1 (Akgul et al., Mol. Life Sci.
57:684-691, 2000), Mac1 Wu et al., Genbank Accession AF059715,
1999), Inohara, N., Gourley, T. S., Carrio, R., Muniz, M., Merino,
J., Garcia, I., Koseki, T., Hu, Y., Chen, S. and Nunez, G., Diva, a
Bcl-2 homologue that binds directly to Apaf-1 and induces
BH3-independent cell death, J. Biol. Chem. 273 (49), 32479-32486
(1998), Boo (Inohara et al., J. Biol. Chem. 273:32479-32486, 1998),
Bax-inhibitors (Kawai et al., FEBS Lett. 464:143-147, 1999), or IAP
(inhibitor of apoptosis, see Clem et al., Trends in Cell Biol.
7:337-339; Liston et al., Nature 379:349-353, 1996; Crook et al.,
Journ. Vir. 67(4):2168-2174, 1993) would reduce the tendency of
recently transformed cells to undergo programmed cell death, and in
the process increase transgene integration and overall
transformation frequencies. Using constructs designed to diminish
the expression of activity of such apoptosis stimulatory genes as
APAF-1, bad (Yang et al., Cell 80:285-291, 1995), bax (Han et al.,
Genes Dev. 10:461-477, 1996), APAF-1/CED-4 (Cecconi et al., Cell
94:727-737, 1998), caspase-3 (Fernandes-Alnemri et al., J. Biol.
Chem. 269:30761-30764, 1994) would have a similar positive effect
on growth enhancement and transformation, all of which are
incorporated herein by reference.
[0034] Other genes useful to the invention include the Kn1 family
of genes (Vollbrecht et al., Nature 350:241-243, 1991; Sentoku et
al., Develop. Biol. 220:358-364, 2000), WUSCHEL (Mayer et al., Cell
95:805-815, 1998), Zwille (Moussian et al., EMBO J. 17:1799-1805,
1998), Aintegumenta (Mizukami et al., PNAS 97:942-947, 2000),
prolifera (Springer et al., Science 268:877-880, 1995), PICKLE
(Ogas et al., PNAS 96:13839-13844, 1999), and FIE (Ohas et al.,
Plant Cell 11:407-416, 1999, all of which are incorporated herein
by reference.
[0035] Other polynucleotides suitable for use in the growth
stimulation vector include the following polynucleotides. Wee1
polynucleotides are disclosed in US99/30957 filed Dec. 21, 1999.
Lec1 polynucleotides are disclosed in US99/26514 filed Nov. 9,
1999. Cyclin D polynucleotides are disclosed in WO 00/17364
published Mar. 30, 2000. CKS polynucleotides are found in 99/61619
filed May 19, 1999. DP polynucleotides are found in Ser. No.
09/503,139 filed Feb. 11, 2000. Cyclin E polynucleotides are found
in Ser. No. 09/496,444 filed Feb. 2, 2000. The disclosures of these
items are incorporated herein by reference.
[0036] Examples of suitable plant virus replicase polynucleotide
sources include wheat dwarf virus, maize streak virus, tobacco
yellow dwarf virus, tomato golden mosaic virus, abutilon mosaic
virus, cassaya mosaic virus, beet curly top virus, bean dwarf
mosaic virus, bean golden mosaic virus, chloris striate mosaic
virus, digitaria streak virus, miscanthus streak virus, maize
streak virus, panicum streak virus, potato yellow mosaic virus,
squash leaf curl virus, sugarcane streak virus, tomato golden
mosaic virus, tomato leaf curl virus, tomato mottle virus, tobacco
yellow dwarf virus, tomato yellow leaf curl virus, African cassaya
mosaic virus, and the bean yellow dwarf virus.
[0037] Replicase from the wheat dwarf virus has been sequenced and
functionally characterized. Replicase binds to a well-characterized
binding motif on the Rb protein (Xie et al., The EMBO Journal Vol.
14 no. 16 pp. 4073-4082, 1995; Orozco et al., Journal of Biological
Chemistry, Vol. 272, No. 15, pp. 9840-9846, 1997; Timmermans et
al., Annual Review Plant Physiology. Plant Mol. Biol. 45:79-112,
1994; Stanley, Genetics and Development 3:91-96, 1996; Davies et
al., Geminivirus Genomes, Chapter 2, and Gutierrez, Plant Biology
1:492-497, 1998). The disclosures of these items are incorporated
herein by reference.
[0038] Other polynucleotides suitable for use in the growth
stimulation vector include viral cell cycle modulator proteins such
as CLINK (Aronson et al. Journal of Virology 74:2968-2972, 2000).
The disclosure of which is incorporated herein by reference.
Examples of other viral sources for this type of protein include
banana bunchy top virus, milk vetch dwarf virus, subterranean color
stunt virus Ageratum yellow vein virus and other representatives of
plant nanoviruses.
[0039] Repressors of plant developmental transitions such as Pickle
and WD polycomb genes including FIE and Medea can be used in the
practice of the invention (Ohad et al., Plant Cell, 1999 Mar., 11
(3):407-416; Plant Cell 1999 May 11(3):765-768; Curr Biol Jan. 27,
2000:10(2)R71-74; Curr Biol Jul. 2, 1998:8(14)R480-484; Science
Apr. 17, 1998:280 (5362) 446-450), all of which are incorporated
herein by reference.
[0040] The growth stimulation polynucleotides can be attached to a
vector, adapter, promoter, transit peptide or linker for cloning
and/or expression of a polynucleotide suitable for use in the
present invention. Additional sequences may be added to such
cloning and/or expression sequences to optimize their function in
cloning and/or expression, to aid in isolation of the
polynucleotide, or to improve the introduction of the
polynucleotide into a cell. Use of cloning vectors, expression
vectors, adapters, and linkers is well known and extensively
described in the art. For a description of such nucleic acids see,
for example, Stratagene Cloning Systems, Catalogs 1995, 1996, 1997
(La Jolla, Calif.); and, Amersham Life Sciences, Inc, Catalog '97
(Arlington Heights, Ill.).
[0041] The construction of expression cassettes that can be
employed in conjunction with the present invention is well known to
those of skill in the art in light of the present disclosure. See,
e.g., Sambrook et al.; Molecular Cloning: A Laboratory Manual; Cold
Spring Harbor, N.Y.; (1989); Gelvin et al.; Plant Molecular Biology
Manual; (1990); Plant Biotechnology: Commercial Prospects and
Problems, eds. Prakash et al.; Oxford & IBH Publishing Co.; New
Delhi, India; (1993); and Heslot, et al.; Molecular Biology and
Genetic Engineering of Yeasts; CRC Press, Inc., USA; (1992); each
incorporated herein in its entirety by reference.
[0042] For example, expression cassettes may include (1) a growth
stimulation polynucleotide under the transcriptional control of 5'
and 3' regulatory sequences and (2) a dominant selectable marker.
Such expression cassettes may also contain, if desired, a promoter
regulatory region (e.g., one conferring inducible, constitutive,
environmentally- or developmentally-regulated, or cell- or
tissue-specific/selective expression), a transcription initiation
start site, a ribosome binding site, an RNA processing signal, a
transcription termination site, and/or a polyadenylation
signal.
[0043] Constitutive, tissue-preferred or inducible promoters can be
employed. Examples of constitutive promoters include the
cauliflower mosaic virus (CaMV) 35S transcription initiation
region, the 1'- or 2'-promoter derived from T-DNA of Agrobacterium
tumefaciens, the ubiquitin 1 promoter (Christensen et al., Plant
Mol. Biol 18:675-689, 1992), the Smas promoter (REF), the cinnamyl
alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos
promoter (Shaw et al., Nucl. Acids Res. 12:7831-7846, 1984), the
pEmu promoter (Last et al, Theor. Applied Genet. 81:581-588, 1991),
the rubisco promoter (Gittins et al., Planta 210:232-240, 2000),
the GRP1-8 promoter and other transcription initiation regions from
various plant genes known to those of skill.
[0044] Examples of inducible promoters are the Adh1 promoter which
is inducible by hypoxia or cold stress (Walker et al., PNAS
84:6624-6628, 1987), the Hsp70 promoter which is inducible by heat
stress (Rochester et al., EMBO J. 5:451-458, 1986), and the PPDK
promoter which is inducible by light (Nomura et al., Plant J.
22:211-221, 2000). Also useful are promoters that are chemically
inducible.
[0045] Examples of promoters under developmental control include
promoters that initiate transcription preferentially in certain
tissues, such as leaves, roots, fruit, seeds, or flowers. An
exemplary promoter is the anther specific promoter 5126 (U.S. Pat.
Nos. 5,689,049 and 5,689,051). Examples of seed-preferred promoters
include, but are not limited to, 27 kD gamma zein promoter and waxy
promoter, Boronat, A., Martinez, M. C., Reina, M., Puigdomenech, P.
and Palau, J.; Isolation and sequencing of a 28 kD glutelin-2 gene
from maize: Common elements in the 5' flanking regions among zein
and glutelin genes; Plant Sci. 47:95-102 (1986) and Reina, M.,
Ponte, I., Guillen, P., Boronat, A. and Palau, J., Sequence
analysis of a genomic clone encoding a Zc2 protein from Zea mays
W64 A, Nucleic Acids Res. 18(21):6426 (1990). See the following
site relating to the waxy promoter: Kloesgen, R. B., Gierl, A.,
Schwarz-Sommer, Z S. and Saedler, H., Molecular analysis of the
waxy locus of Zea mays, Mol. Gen. Genet. 203:237-244 (1986).
Promoters that express in the embryo, pericarp, and endosperm are
disclosed in U.S. application Ser. Nos. 60/097,233 filed Aug. 20,
1998 and 60/098,230 filed Aug. 28, 1998. The disclosures each of
these are incorporated herein by reference in their entirety.
[0046] Either heterologous or non-heterologous (i.e., endogenous)
promoters can be employed to direct expression of the nucleic acids
of the present invention. These promoters can also be used, for
example, in expression cassettes to drive expression of antisense
nucleic acids to reduce, increase, or alter concentration and/or
composition of the proteins of the present invention in a desired
tissue.
[0047] If polypeptide expression is desired, it is generally
desirable to include a polyadenylation region at the 3'-end of a
polynucleotide coding region. The polyadenylation region can be
derived from the natural gene, from a variety of other plant genes,
or from T-DNA. The 3' end sequence to be added can be derived from,
for example, the nopaline synthase or octopine synthase genes, or
alternatively from another plant gene, or less preferably from any
other eukaryotic gene.
[0048] An intron sequence can be added to the 5' untranslated
region or the coding sequence of the partial coding sequence to
increase the amount of the mature message that accumulates. See for
example Buchman and Berg, Mol. Cell Biol. 8: 4395-4405 (1988);
Callis et al., Genes Dev. 1:1183-1200 (1987). Use of maize introns
Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the
art. See generally, The Maize Handbook, Chapter 116, Freeling and
Walbot, Eds., Springer, New York (1994).
[0049] The vector comprising the polynucleotide sequences useful in
the present invention may comprise a marker gene that confers a
selectable phenotype on plant cells. Usually, the selectable marker
gene will encode antibiotic or herbicide resistance. Suitable genes
include those coding for resistance to the antibiotic spectinomycin
or streptomycin (e.g., the aada gene), the streptomycin
phosphotransferase (SPT) gene coding for streptomycin resistance,
the neomycin phosphotransferase (NPTII) gene encoding kanamycin or
geneticin resistance, the hygromycin phosphotransferase (HPT) gene
coding for hygromycin resistance.
[0050] Suitable genes coding for resistance to herbicides include
those which act to inhibit the action of acetolactate synthase
(ALS), in particular the sulfonylurea-type herbicides (e.g., the
acetolactate synthase (ALS) gene containing mutations leading to
such resistance in particular the S4 and/or Hra mutations), those
which act to inhibit action of glutamine synthase, such as
phosphinothricin or basta (e.g., the bar gene), or other such genes
known in the art. The bar gene encodes resistance to the herbicide
basta and the ALS gene encodes resistance to the herbicide
chlorsulfuron.
[0051] Typical vectors useful for expression of nucleic acids in
higher plants are well known in the art and include vectors derived
from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens
described by Rogers et al., Meth. In Enzymol., 153:253-277 (1987).
Exemplary A. tumefaciens vectors useful herein are plasmids pKYLX6
and pKYLX7 of Schardl et al., Gene 61:1-11 (1987) and Berger et
al., Proc. Natl. Acad. Sci. U.S.A. 86:8402-8406 (1989). Another
useful vector herein is plasmid pBI101.2 that is available from
Clontech Laboratories, Inc. (Palo Alto, Calif.). A variety of plant
viruses that can be employed as vectors are known in the art and
include cauliflower mosaic virus (CaMV), geminivirus, brome mosaic
virus, and tobacco mosaic virus.
[0052] The growth stimulation polynucleotide can be expressed in
either sense or anti-sense orientation as desired. In plant cells,
it has been shown that antisense RNA inhibits gene expression by
preventing the accumulation of mRNA which encodes the enzyme of
interest, see, e.g., Sheehy et al., Proc. Nat'l. Acad. Sci. (USA)
85:8805-8809 (1988); and Hiatt et al., U.S. Pat. No. 4,801,340.
[0053] Another method of suppression is sense suppression. For an
example of the use of this method to modulate expression of
endogenous genes see, Napoli et al., The Plant Cell 2:279-289
(1990) and U.S. Pat. No. 5,034,323. Another method of
down-regulation of the protein involves using PEST sequences that
provide a target for degradation of the protein. Still other
methods of suppression are disclosed in WO 99/53050, which
discloses a method that involves both sense and antisense
suppression, i.e. hairpin technology.
[0054] Catalytic RNA molecules or ribozymes can also be used to
inhibit expression of plant genes. The inclusion of ribozyme
sequences within antisense RNAs confers RNA-cleaving activity upon
them, thereby increasing the activity of the constructs. The design
and use of target RNA-specific ribozymes is described in Haseloff
et al., Nature 334: 585-591 (1988).
[0055] The methods of the present invention can be used with any
cell such as bacteria, yeast, insect, non-human mammalian, or
preferably plant cells.
[0056] Typically, an intermediate host cell will be used in the
practice of this invention to increase the copy number of the
cloning vector. With an increased copy number, the vector
containing the nucleic acid of interest can be isolated in
significant quantities for introduction into the desired plant
cells. Host cells that can be used in the practice of this
invention include prokaryotes, including bacterial hosts such as
Eschericia coli, Salmonella typhimurium, and Serratia marcescens.
Eukaryotic hosts such as yeast or filamentous fungi may also be
used in this invention. It preferred to use plant promoters that do
not cause expression of the polypeptide in bacteria.
[0057] Commonly used prokaryotic control sequences include
promoters such as the beta lactamase (penicillinase) and lactose
(lac) promoter systems (Chang et al., Nature 198:1056 (1977)), the
tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids
Res. 8:4057 (1980)) and the lambda derived P L promoter and N-gene
ribosome binding site (Shimatake et al., Nature 292:128 (1981)).
The inclusion of selection markers in DNA vectors transfected in E.
coli is also useful. Examples of such markers include genes
specifying resistance to ampicillin, tetracycline, or
chloramphenicol.
[0058] The vector is selected to allow introduction into the
appropriate host cell. Bacterial vectors are typically of plasmid
or phage origin. Expression systems for expressing a protein of the
present invention are available using Bacillus sp. and Salmonella
(Palva et al., Gene 22:229-235 (1983); Mosbach et al., Nature
302:543-545 (1983)).
[0059] The invention can be practiced in a wide range of plants
such as monocots or dicots. For example, the methods of the present
invention can be employed in corn, soybean, sunflower, safflower,
potato, tomato, sorghum, canola, wheat, alfalfa, cotton, rice,
barley and millet.
[0060] The method of transformation is not critical to the
invention; various methods of transformation are currently
available. As newer methods are available to transform host cells
they may be directly applied. Accordingly, a wide variety of
methods have been developed to insert a DNA sequence into the
genome of a host cell to obtain the transcription and/or
translation of the sequence. Thus, any method that provides for
efficient transformation/transfection may be employed.
[0061] A DNA sequence coding for the desired polynucleotide useful
in the present invention, for example a cDNA, RNA or a genomic
sequence, will be used to construct an expression cassette that can
be introduced into the desired host cell. Isolated nucleic acid
acids useful in the present invention can be introduced into plants
according techniques known in the art. Generally, expression
cassettes as described above and suitable for transformation of
plant cells are prepared.
[0062] Methods for transforming various host cells are disclosed in
Klein et al. "Transformation of microbes, plants and animals by
particle bombardment", Bio/Technol. New York, N.Y., Nature
Publishing Company, March 1992, v. 10 (3) pp. 286-291. Techniques
for transforming a wide variety of higher plant species are well
known and described in the technical, scientific, and patent
literature. See, for example, Weising et al., Ann. Rev. Genet.
22:421-477 (1988).
[0063] For example, the DNA construct may be introduced directly
into the genomic DNA of the plant cell using techniques such as
electroporation, PEG-mediated transfection, particle bombardment,
silicon fiber delivery, or microinjection of plant cell protoplasts
or embryogenic callus. See, e.g., Tomes et al., Direct DNA Transfer
into Intact Plant Cells Via Microprojectile Bombardment. pp.
197-213 in Plant Cell, Tissue and Organ Culture, Fundamental
Methods. eds. O. L. Gamborg and G. C. Phillips. Springer-Verlag
Berlin Heidelberg New York, 1995. The introduction of DNA
constructs using polyethylene glycol precipitation is described in
Paszkowski et al., Embo J. 3:2717-2722 (1984). Electroporation
techniques are described in Fromm et al., Proc. Natl. Acad. Sci.
82:5824 (1985). Ballistic transformation techniques are described
in Klein et al., Nature 327:70-73 (1987).
[0064] Alternatively, the DNA constructs may be combined with
suitable T-DNA flanking regions and introduced into a conventional
Agrobacterium tumefaciens host vector. The virulence functions of
the Agrobacterium tumefaciens host will direct the insertion of the
construct and adjacent marker into the plant cell DNA when the cell
is infected by the bacteria. Agrobacterium tumefaciens-meditated
transformation techniques are well described in the scientific
literature. See, for example Horsch et al., Science 233:496-498
(1984), and Fraley et al., Proc. Natl. Acad. Sci. 80:4803 (1983).
For instance, Agrobacterium transformation of maize is described in
U.S. Pat. No. 5,981,840. Agrobacterium transformation of monocot is
found in U.S. Pat. No. 5,591,616. Agrobacterium transformation of
soybeans is described in U.S. Pat. No. 5,563,055.
[0065] Other methods of transformation include (1) Agrobacterium
rhizogenes-mediated transformation (see, e.g., Lichtenstein and
Fuller In: Genetic Engineering, vol. 6, P W J Rigby, Ed., London,
Academic Press, 1987; and Lichtenstein, C. P., and Draper, J, In:
DNA Cloning, Vol. 11, D. M. Glover, Ed., Oxford, IRI Press, 1985),
Application PCT/US87/02512 (WO 88/02405 published Apr. 7, 1988)
describes the use of A. rhizogenes strain A4 and its Ri plasmid
along with A. tumefaciens vectors pARC8 or pARC16 (2)
liposome-mediated DNA uptake (see, e.g., Freeman et al., Plant Cell
Physiol. 25:1353, 1984), (3) the vortexing method (see, e.g.,
Kindle, Proc. Natl. Acad. Sci., USA 87:1228, (1990).
[0066] DNA can also be introduced into plants by direct DNA
transfer into pollen as described by Zhou et al., Methods in
Enzymology, 101:433 (1983); D. Hess, Intern Rev. Cytol., 107:367
(1987); Luo et al., Plane Mol. Biol. Reporter 6:165 (1988).
Expression of polypeptide coding nucleic acids can be obtained by
injection of the DNA into reproductive organs of a plant as
described by Pena et al., Nature 325:274 (1987). DNA can also be
injected directly into the cells of immature embryos and the
rehydration of desiccated embryos as described by Neuhaus et al.,
Theor. Appl. Genet. 75:30 (1987); and Benbrook et al., in
Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54
(1986).
[0067] Animal and lower eukaryotic (e.g., yeast) host cells are
competent or rendered competent for transformation by various
means. There are several well-known methods of introducing DNA into
animal cells. These include: calcium phosphate precipitation,
fusion of the recipient cells with bacterial protoplasts containing
the DNA, treatment of the recipient cells with liposomes containing
the DNA, DEAE dextran, electroporation, biolistics, and
micro-injection of the DNA directly into the cells. The transfected
cells are cultured by means well known in the art. Kuchler, R. J.,
Biochemical Methods in Cell Culture and Virology, Dowden,
Hutchinson and Ross, Inc. (1977).
[0068] Transformed plant cells which are derived by any of the
above transformation techniques can be cultured to regenerate a
whole plant which possesses the transformed genotype. Such
regeneration techniques often rely on manipulation of certain
phytohormones in a tissue culture growth medium, typically relying
on a biocide and/or herbicide marker which has been introduced
together with a polynucleotide of the present invention. For
transformation and regeneration of maize see, Gordon-Kamm et al.,
The Plant Cell, 2:603-618 (1990).
[0069] Once the responsive target cell is transformed with the
growth stimulation polynucleotide, it is re-transformed with a gene
of interest. The transformed cell can be from transformed callus,
transformed embryo, T0 regenerated plants or its parts, progeny of
T0 plants or parts thereof as long as the growth stimulation
polynucleotide is present.
[0070] Genes of interest can include any gene, generally, those
involved in oil, starch, protein, carbohydrate or nutrient
metabolism as well as those affecting kernel size, sucrose loading,
and the like. The gene of interest may be involved in regulating
the influx of nutrients, disease resistance and in regulating
expression of phytate genes particularly to lower phytate levels in
the seed.
[0071] General categories of genes of interest for the purpose of
present invention include for example, those genes involved in
information, such as Zinc fingers, those involved in communication,
such as kinases, and those involved in housekeeping, such as heat
shock proteins. More specific categories of transgenes, for
example, include genes encoding important traits for agronomics,
insect resistance, disease resistance, herbicide resistance, and
grain characteristics. It is recognized that any gene of interest
can be operably linked to the promoter of the invention and
expressed in the seed.
[0072] Important traits such as oil, starch and protein content can
be genetically altered. Modifications include altering the content
of oleic acid, saturated and unsaturated oils, increasing levels of
lysine and sulfur-containing amino acids and providing other
essential amino acids, and also modification of starch and
cellulose. Hordothionin protein modifications are described in
WO94/16078; WO96/38562; WO96/08220; and U.S. Pat. No. 5,703,409
issued Dec. 30, 1997 the disclosures of which are incorporated
herein in their entirety by reference. Another example is lysine
and/or sulfur rich seed protein encoded by the soybean 2S albumin
described in WO97/35023, and the chymotrypsin inhibitor from
barley, Williamson et al. (1987) Eur. J. Biochem. 165:99-106, the
disclosures of each are incorporated by reference.
[0073] Derivatives of the following genes can be made by site
directed mutagenesis to increase the level of preselected amino
acids in the encoded polypeptide. For example, the gene encoding
the barley high lysine polypeptide (BHL) is derived from barley
chymotrypsin inhibitor, WO98/20133, incorporated herein by
reference. Other proteins include methionine-rich plant proteins
such as from sunflower seed (Lilley et al. (1989) Proceedings of
the World Congress on Vegetable Protein Utilization in Human Foods
and Animal Feedstuffs; Applewhite, H. (ed.); American Oil Chemists
Soc., Champaign, Ill.: pp. 497-502, incorporated herein in its
entirety by reference), corn (Pedersen et al. (1986) J. Biol. Chem.
261:6279; Kirihara et al. (1988) Gene 71:359, both incorporated
herein in its entirety by reference) and rice (Musumura et al.
(1989) Plant Mol. Biol. 12:123, incorporated herein in its entirety
by reference). Other agronomically important genes encode Floury 2,
growth factors, seed storage factors and transcription factors.
[0074] Commercial traits can also be encoded on a gene(s) which
could alter or increase for example, starch for the production of
paper, textiles, and ethanol, or provide expression of proteins
with other commercial uses. Another important commercial use of
transformed plants is the production of polymers and bioplastics
such as described in U.S. Pat. No. 5,602,321 issued Feb. 11, 1997.
Genes such as B-ketothiolase, PHBase (polyhydroxyburyrate synthase)
and acetoacetyl-CoA reductase (see Schubert et al. (1988) J.
Bacteriol 170(12):5837-5847) facilitate expression of
polyhyroxyalkanoates (PHAs).
[0075] Exogenous products include plant enzymes and products as
well as those from other sources including prokaryotes and other
eukaryotes. Such products include enzymes, cofactors, hormones, and
the like. The level of seed proteins, particularly modified seed
proteins having improved amino acid distribution to improve the
nutrient value of the seed can be increased. This is achieved by
the expression of such proteins having enhanced amino acid
content.
[0076] Insect resistance genes may encode resistance to pests that
have great yield drag such as rootworm, cutworm, European Corn
Borer, and the like. Such genes include, for example, Bacillus
thuringiensis endotoxin genes (U.S. Pat. Nos. 5,366,892; 5,747,450;
5,737,514; 5,723,756; 5,593,881; Geiser et al. (1986) Gene 48:109);
lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825); and the
like.
[0077] Genes encoding disease resistance traits may include
detoxification genes, such as against fumonosin (U.S. patent
application Ser. No. 08/484,815 filed Jun. 7, 1995); avirulence
(avr) and disease resistance (R) genes (Jones et al. (1994) Science
266:789; Martin et al. (1993) Science 262:1432; Mindrinos et al.
(1994) Cell 78:1089; and the like.
[0078] Agronomic traits in seeds can be improved by altering
expression of genes that affect the response of seed growth and
development during environmental stress, Cheikh-N et al. (1994)
Plant Physiol. 106(1):45-51) and genes controlling carbohydrate
metabolism to reduce kernel abortion in maize, Zinselmeier et al.
(1995) Plant Physiol. 107(2):385-391.
[0079] The gene of interest or the growth stimulation
polynucleotide may be an antisense sequence for a targeted gene. By
"antisense DNA nucleotide sequence" is intended a sequence that is
in inverse orientation to the 5'-to-3' normal orientation of that
nucleotide sequence. When delivered into a plant cell, expression
of the antisense DNA sequence prevents normal expression of the DNA
nucleotide sequence for the targeted gene. The antisense nucleotide
sequence encodes an RNA transcript that is complementary to and
capable of hybridizing to the endogenous messenger RNA (mRNA)
produced by transcription of the DNA nucleotide sequence for the
targeted gene. In this case, production of the native protein
encoded by the targeted gene is inhibited to achieve a desired
response. Thus the promoter sequences disclosed herein may be
operably linked to antisense DNA sequences to reduce or inhibit
expression of a native protein in the plant seed.
[0080] Transformed plants cells can be regenerated, e.g., from
single cells, callus tissue or leaf discs according to standard
plant tissue culture techniques. It is well known in the art that
various cells, tissues, and organs from almost any plant can be
successfully cultured to regenerate an entire plant. Plant
regeneration from cultured protoplasts is described in Evans et
al., Protoplasts Isolation and Culture, Handbook of Plant Cell
Culture, Macmillan Publishing Company, New York, pp. 124-176
(1983); and Binding, Regeneration of Plants, Plant Protoplasts, CRC
Press, Boca Raton, pp. 21-73 (1985).
[0081] The regeneration of plants containing the foreign gene
introduced by Agrobacterium can be achieved as described by Horsch
et al., Science, 227:1229-1231 (1985) and Fraley et al., Proc.
Natl. Acad. Sci. U.S.A., 80:4803 (1983). This procedure typically
produces shoots within two to four weeks and these transformant
shoots are then transferred to an appropriate root-inducing medium
containing the selective agent and an antibiotic to prevent
bacterial growth. Transgenic plants of the present invention may be
fertile or sterile.
[0082] Regeneration can also be obtained from plant callus,
explants, organs, or parts thereof. Such regeneration techniques
are described generally in Klee et al., Ann. Rev. of Plant Phys.
38: 467-486 (1987). The regeneration of plants from either single
plant protoplasts or various explants is well known in the art.
See, for example, Methods for Plant Molecular Biology, A. Weissbach
and H. Weissbach, eds., Academic Press, Inc., San Diego, Calif.
(1988). For maize cell culture and regeneration see generally, The
Maize Handbook, Freeling and Walbot, Eds., Springer, New York
(1994); Corn and Corn Improvement, 3.sup.rd edition, Sprague and
Dudley Eds., American Society of Agronomy, Madison, Wis.
(1988).
[0083] One of skill will recognize that after the expression
cassette is stably incorporated in transgenic plants and confirmed
to be operable, it can be introduced into other plants by sexual
crossing. Any of a number of standard breeding techniques can be
used, depending upon the species to be crossed.
[0084] In vegetatively propagated crops, mature transgenic plants
can be propagated by the taking of cuttings or by tissue culture
techniques to produce multiple identical plants. Selection of
desirable transgenics is made and new varieties are obtained and
propagated vegetatively for commercial use. In seed propagated
crops, mature transgenic plants can be self-crossed to produce a
homozygous inbred plant. The inbred plant produces seed containing
the newly introduced heterologous nucleic acid. These seeds can be
grown to produce plants that would produce the selected
phenotype.
[0085] Parts obtained from the regenerated plant, such as flowers,
seeds, leaves, branches, fruit, and the like are included in the
invention. Progeny and variants, and mutants of the regenerated
plants are also included within the scope of the invention,
provided that these parts comprise the introduced nucleic acid
sequences.
[0086] Transgenic plants expressing a selectable marker can be
screened for transmission of the gene(s) of interest, for example,
standard immunoblot and DNA detection techniques. Transgenic lines
are also typically evaluated on levels of expression of the
heterologous nucleic acid. Expression at the RNA level can be
determined initially to identify and quantitate expression-positive
plants. Standard techniques for RNA analysis can be employed and
include PCR amplification assays using oligonucleotide primers
designed to amplify only the heterologous RNA templates and
solution hybridization assays using heterologous nucleic
acid-specific probes.
[0087] The RNA-positive plants can then be analyzed for protein
expression by Western immunoblot analysis using the specifically
reactive antibodies of the present invention. In addition, in situ
hybridization and immunocytochemistry according to standard
protocols can be done using heterologous nucleic acid specific
polynucleotide probes and antibodies, respectively, to localize
sites of expression within transgenic tissue. Generally, a number
of transgenic lines are usually screened for the incorporated
nucleic acid to identify and select plants with the most
appropriate expression profiles.
[0088] Seeds derived from plants regenerated from re-transformed
plant cells, plant parts or plant tissues, or progeny derived from
the regenerated plants, may be used directly as feed or food, or
further processing may occur.
[0089] Through the integration of a gene or genes into the elite
(recalcitrant) maize-inbreds whose stable expression might have a
positive influence on transformation, the following data
demonstrate potential in increasing the overall genetic
transformation throughput of elite maize germplasm. It is expected
that integration for re-transformation with other genes or gene
combinations will further improve the elite inbred transformation
frequency.
[0090] The present invention will be further described by reference
to the following detailed examples. It is understood, however, that
there are many extensions, variations, and modifications on the
basic theme of the present invention beyond that shown in the
examples and description, which are within the spirit and scope of
the present invention.
[0091] All publications, patents, and patent applications cited
herein are hereby incorporated by reference.
EXAMPLES
Example 1
DNA Delivery Methods
[0092] Transformation of the Lec1 plasmids, PHP16102, PHP16215, and
PHP16273 along with the expression cassette UBI::moPAT-GPFm::pinII
into genotype Hi-II followed a well-established bombardment
transformation protocol used for introducing DNA into the scutellum
of immature maize embryos (Songstad, D. D. et al., In Vitro Cell
Dev. Biol. Plant 32:179-183, 1996). It is noted that any suitable
method of transformation can be used, such as
Agrobacterium-mediated transformation and many other methods. Cells
were transformed by culturing maize immature embryos (approximately
1-1.5 mm in length) onto medium containing N6 salts, Erikkson's
vitamins, 0.69 g/l proline, 2 mg/l 2,4-D and 3% sucrose. After 4-5
days of incubation in the dark at 28.degree. C., embryos were
removed from the first medium and cultured onto similar medium
containing 12% sucrose. Embryos were allowed to-:acclimate to this
medium for 3 h prior to transformation. The scutellar surface of
the immature embryos was targeted using particle bombardment.
Embryos were transformed using the PDS-1000 Helium Gun from Bio-Rad
at one shot per sample using 650 PSI rupture disks. DNA delivered
per shot averaged at 0.1667% g. Following bombardment, all embryos
were maintained on standard maize culture medium (N6 salts,
Erikkson's vitamins, 0.69 g/l proline, 2 mg/l 2,4-D, 3% sucrose)
for 2-3 days and then transferred to N6-based medium containing 3
mg/L Bialaphos.RTM.. Plates were maintained at 28.degree. C. in the
dark and were observed for colony recovery with transfers to fresh
medium every two to three weeks. Recovered colonies and plants were
scored based on GFP visual expression, leaf painting sensitivity to
a 1% application of Ignite.RTM. herbicide, and molecular
characterization via PCR and Southern analysis.
[0093] Transformation of the RepA containing plasmid (PHP15524) and
control plasmid (PHP15325) into Pioneer Hi-Bred International, Inc.
proprietary maize inbreds N46 and P38 were done using the
Agrobacterium mediated DNA delivery method, as described by U.S.
Pat. No. 5,981,840 with the following modifications. It is noted
that any suitable method of transformation can be used, such as
particle-mediated transformation, as well as many other methods.
Agrobacteria were grown to the log phase in liquid minimal A medium
containing 100 .mu.M spectinomycin. Embryos were immersed in a log
phase suspension of Agrobacteria adjusted to obtain an effective
concentration of 5.times.10.sup.8 cfu/ml. Embryos were infected for
5 minutes and then co-cultured on culture medium containing
acetosyringone for 7 days at 20.degree. C. in the dark. After 7
days, the embryos were transferred to standard culture medium (MS
salts with N6 macronutrients, 1 mg/L 2,4-D, 1 mg/L Dicamba, 20 g/L
sucrose, 0.6 g/L glucose, 1 mg/L silver nitrate, and 100 mg/L
carbenicillin) with 3 mg/L Bialaphos.RTM. as the selective agent.
Plates were maintained at 28.degree. C. in the dark and were
observed for colony recovery with transfers to fresh medium every
two to three weeks. Recovered colonies and plants were scored based
on GFP visual expression, leaf painting sensitivity to a 1%
application of Ignite.RTM. herbicide, and molecular
characterization via PCR and Southern analysis.
Example 2
Re-Transformation of RepA Transgenic Progeny Results in Increased
Transformation Frequency in Elite Maize Inbreds
[0094] The plasmids listed in the table below were used to evaluate
the influence of RepA on stable expression of co-delivered
transgenes. Two vectors were constructed to test the repA gene
constructs using Agrobacterium-mediated transformation. A control
vector, designated PHP15303, carried two gene cassettes. The first
comprised a ubiquitin promoter:intron sequence driving a Green
Fluorescent Protein (GFP) coding sequence. This coding sequence had
previously been modified to optimize codons for expression in maize
and to include an intron (precluding expression of the GFP in
bacterial cells). A polyadenylation signal sequence from the pinII
gene was used. The second gene in this vector was the selectable
marker CaMV35S Enhancer:CaMV35S promoter:Omega Prime 5'UTR:ADH1
intron1: BAR:pinII. This control vector was mated into
Agrobacterium tumefaciens LBA4404 carrying a superbinary vir
plasmid (PHP10523). The resulting 15303/10523 cointegrate plasmid
was designated PHP15325.
[0095] The second vector was derived from the wheat dwarf virus
(WDV) promoter:replicase gene originally obtained from Jo Messing
(pWI-11). The myb region of the rep Exon 2 was deleted (as a 130 bp
Asp700 fragment) to create plasmid PHP14807 (WDV
promoter:REP-EXON1:REP-INTRON1:REP-EXON2 (Asp700 DELETION):WDV
TERM). This expression cassette was cloned into a polylinker in an
intermediate vector to pick up flanking BstEII sites. The cassette
was then moved as a 1.93 kb BstEII fragment into compatible BstEII
sites in PHP15303 just upstream of the two genes described above.
This three-gene plasmid was designated PHP15440. After mating into
Agrobacterium tumefaciens LBA4404 carrying a superbinary vir
plasmid (PHP10523) as above, the final 15440/10523 cointegrate
plasmid was designated PHP15524.
1TABLE I Plasmid Description P15325 RB/e35S::BAR::PinII + Ubi::Ubi
intron::GFPm::PinII/LB P15524 RB/e35S::BAR::PinII + Ubi::Ubi
intron::GFPm::PinII + wdv LIR::RepA(ASP700)/LB
[0096] The visible marker gene GFP (green fluorescence protein;
Chalfie et al., Science 263:802, 1994) has been described as has
the maize-optimized GFP (GFPm; see copending US Patent Application
WO 97/41228). The Ubiquitin promoter has been described
(Christensen et al., Plant Mol. Biol. 12:619-623 (1989) and
Christensen et al., Plant Mol. Biol. 18:675-689 (1992), as has the
pinII (An et al., 1989, Plant Cell 1:115-122) 3' region used in
these cassettes.
[0097] Transformations of the RepA containing plasmid (P15524) and
control plasmid (P15325) in maize inbreds P38 and N46 were done
using the Agrobacterium mediated DNA delivery method, as described
by U.S. Pat. No. 5,981,840 with modifications as listed in Example
1. Embryos were co-cultured on culture medium with acetosyringone
for 7 days at 20.degree. C. After 7 days the embryos were
transferred to standard culture medium containing 3 mg/L Bialaphos
with the addition of 100 mg/L carbenicillin to kill off residual
Agrobacteria. Total embryos cultured per ear were divided between
the two plasmids to evaluate the effect of RepA on inbred
transformation. Fertile plants with normal phenotypes were
recovered based on reporter gene expression, leaf resistance to
herbicide, and molecular analyses in both RepA events and in
control events containing only BAR and GFPm.
[0098] A study was initiated to evaluate if the integrated RepA
transgene from these events would have any effect on the frequency
of subsequent transformations. T.sub.1 embryos from both RepA and
control events were selected. Ears to be harvested were infused at
4DAP with compounds found to yield optimal embryogenic response
within the genotype [1108P application], harvested at 1 DAP, and
bombarded using the particle gun following the methodology listed
in Example 1. The visual marker CRC was used as the transgene for
this study. The marker was put into vector PHP7951, containing the
nos promoter driving the CRC transgene with a PinII terminator. CRC
has been previously described (Bruce, W. et al., Plant Cell
12:65-79, 2000). CRC expressing sectors were recovered at high
frequencies without selective pressure across independent events
only from the embryos segregating for the RepA transgene (based on
GFPm expression). Wild type segregates as well as control events
containing only the selectable marker and reporter gene did not
yield high frequencies of transformation (Table II). These data
demonstrate that RepA expression improves re-transformation
frequencies.
2TABLE II Maize Elite Inbred Re-Transformation Data Event # #
Overall Segregated SID Genotype DNA # Embryos GRF+ CRC+ Frequency
Frequency RepA Integrated: 1025332 P38 P15524 24 14 1 4.2% 7.1%
1025341 P38 P15524 72 41 14 19.4% 34% 1028139 P38 P15524 21 3 0 0%
0% 1052134 N46 P15524 15 6 4 29% 66% 1038724 N46 P15524 150 1 18
12% 100% 1052136 N46 P15524 42 18 2 5% 11.1% Controls: 1027793 P38
P15325 98 6 0 0 0 1033190 P38 P15325 60 25 0 0 0 1025793 P38 P15325
60 N/A 0 0 0 1045751 P38 P15325 7 5 0 0 0 1025334 P38 P15325 94 38
1 0 0 (died) 1029080 P38 P15325 196 98 1 0.5% 1% 1025723 P38 P15325
160 67 0 0 0 1029082 P38 P15325 30 11 0 0 0 P15524:
RB/e35S::BAR::PinII + Ubi::Ubi intron::GFPm intron::PinII +
wdv::RepA(ASP700)/LB P15325: RB/e35S::BAR::PinII + Ubi::Ubi
intron::GPFm intron::PinII/LB
Example 3
Validation of Re-Transformation of RepA Transgenic Progeny Results
in Increased Transformation Frequency in Elite Maize Inbreds
[0099] To further evaluate the effect of the integrated RepA
transgene on maize inbred transformation, T.sub.1 seed from RepA
(PHP15524) events were planted from genotypes N46 and P38. Wild
type seed were also planted to serve as transformation controls.
The T.sub.1 plants were screened to identify segregates for the
RepA transgene, and outcrossed to their wild type recurrent parent
or other elite inbreds PH24E and PH09B. A group (both T.sub.1 and
controls) of the ears produced were infused at 4DAP (1108P patent
application) with compounds found to yield optimal embryogenic
response within the genotype and harvested for immature embryo
transformation. Transformations were completed using the
Agrobacterium mediated DNA delivery method, as described by U.S.
Pat. No. 5,981,840 with modifications as listed in Example 1. Total
embryos cultured per ear were divided between the plasmids
PHP16543, containing the visual marker CRC, as described above, and
PHP16340, containing the Rice EPSPS gene conferring resistance to
glyphosate, as described in Table III below:
3 TABLE III Plasmid Description PHP16543 RB/NOS-C(G)RC-PinII +
Ubi-moPAT-35S/LB PHP16340 RB/RiceEPSPS pro-eMPU-e35S-RiceEPSPS
intron-Rice EPSPS-Rice EPSPS term/LB
[0100] All embryos were co-cultured on culture medium with
acetosyringone for 7 days at 20.degree. C. After 7 days, the
embryos were transferred to standard culture medium with the
addition of 100 mg/L carbenicillin and either no selective agent
(for transformations done with PHP16543) or 0.25 mM glyphosate (for
transformations done with PHP16340). After 7 weeks, embryos were
evaluated for normal growth on glyphosate selection or stable
anthocyanin expression. Transformation frequencies (to date based
on the embryos available for scoring) are as shown in Table IV
below.
4TABLE IV T1 Cross or Wild Parent Parent # Expressing Overall Type
Control Genotype(s) SID DNA # Embryos Colonies Frequency P38 ear 1
P38 N/A PHP16340 21 0 0% wild type P38 ear 1 P38 N/A PHP16543 21 1
4.8% wild type P38 ear 2 P38 N/A PHP16340 4 0 0% wild type P38 ear
2 P38 N/A PHP16543 2 0 0% wild type PH09B, ear 1 PH09B N/A PHP16340
30 0 0% wild type PH09B, ear 1 PH09B N/A PHP16543 29 0 0% wild type
PH24E, ear 1 PH24E N/A PHP16340 23 0 0% wild type PH24E, ear 1
PH24E N/A PHP16543 31 0 0% wild type PH24E, ear 2 PH24E N/A
PHP16340 23 0 0% wild type PH24E, ear 2 PH24E N/A PHP16543 22 0 0%
wild type PH24E/1133200 PH24E 1023702 PHP16340 40 2 5% and P38
PH24E/1133200 PH24E 1023702 PHP16543 34 2 5.9% and P38 1133131/P38
P38 1025298 PHP16340 32 19 59.4% 1133131/P38 P38 1025298 PHP16543
28 6 21.4% 1133132/P38 P38 1025298 PHP16340 70 5 7.1% 1133132/P38
P38 1025298 PHP16543 82 8 8.04% 1133133/P38 P38 1025298 PHP16340 42
6 14.3% 1133133/P38 P38 1025298 PHP16543 11 1 9.1% 1133161/P38 P38
1025298 PHP16340 19 5 26.3% 1133161/P38 P38 1025298 PHP16543 42 0
0% PH09B/1133137 PH09B 1025298 PHP16340 41 4 9.8% and P38
PH09B/1133137 PH09B 1025298 PHP16543 45 1 2.2% and P38 1133064/N46
N46 1033131 PHP16340 32 2 6.3% 1133064/N46 N46 1033131 PHP16543 41
2 4.9% 1133067/N46 N46 1033131 PHP16340 36 2 5.5% 1133067/N46 N46
1033131 PHP16543 16 0 0% 1133068/N46 N46 1033131 PHP16340 11 4
36.6% 1133068/N46 N46 1033131 PHP16543 16 1 6.3% 1133082/N46 N46
1033131 PHP16340 44 5 11.4% 1133082/N46 N46 1033131 PHP16543 47 2
4.3% 1133103/N46 N46 1033131 PHP16340 9 4 44.4% 1133103/N46 N46
1033131 PHP16543 13 1 7.7%
[0101] The level of glyphosate selection used in this study was
determined to be optimal for eliminating any wild type growth from
maize embryos based on kill curve studies done using ranges of
glyphosate from 0.01 mM to 2 mM. Genotypes P38, PH24E, and PH09B
were evaluated.
[0102] Pending molecular confirmation data for segregation ratios
for RepA and for the newly transformed transgenes, and based on
visual inspection of GPFm expression, it is predicted that the
above transformations will correspond to the data produced in the
previous example; RepA expression improves re-transformation
frequencies in inbreds.
Example 4
Re-Transformation of LEC1-Transgenic Progeny Results in Elevated
Transformation Frequency in Hi-II
[0103] Agrobacterium Mediated Transformation
[0104] As the starting point for Agrobacterium-mediated
re-transformation experiments, regenerated Hi-II T0 transformants
were produced containing maize LEC1 expression cassettes and
UBI::moPAT.about.GFP::pinII. The LEC1 expression cassettes used the
nopaline synthase promoter from Agrobacterium tumefaciens (Shaw et
al., Nucl. Acids Res. 12:7831-7846, 1984) or modified nos promoters
as described below. The PAT.about.GFP cassette contained a
maize-optimized gene encoding phosphinothricin acetyltransferase
(moPAT, see co-Pending Application WO9830701) followed by a
sequence encoding 4.times.(GSSS) to create a flexible polypeptide
linker, and then a maize-optimized nucleic acid sequence encoding
Green Fluorescence Protein (GFP; see co-Pending Application WO
97/41228 published Nov. 6, 1997). This PAT.about.GFP fusion
construct was driven by the maize ubiquitin promoter (Christensen
et al., Plant Mol. Biol. 18:675-689, 1992) and contains a potato
proteinase inhibitor II 3' sequence (An et al., Plant Cell
1:115-122, 1989). Transformants containing
UBI::moPAT.about.GFP::pinII and one of three different LEC1
expression cassettes were tested; with LEC1 being driven by a nos
promoter (PHP16102), a truncated version containing 85 bases of the
nos sequence (PHP16215), or a nos promoter with additional STOP
codons added before the START in order to attenuate expression
(PHP16273).
[0105] Transgenic Hi-II plants containing a co-segregating LEC1
expression cassette and the UBI::PAT.about.GFP expression cassette
were crossed to wild-type (non-transformed) Hi-II plants (using the
non-transformed parent as the pollen donor). As expected from such
a cross, the developing embryos on these ears segregated either for
transgene expression or wild-type. Immature embryos were harvested
12 days after pollination and transformed with an Agrobacterium
binary plasmid containing PHP16449 (UBI::moCAH::pinII, moCAH is a
maize optimized [for codon usage] gene that encodes for the
Myrothecium verrucaria cyanamide hydratase protein [CAH] that can
hydrate cyanamide to non-toxic urea). A standard
Agrobacterium-mediated transformation protocol (U.S. Pat. No.
5,981,840) adapted for cyanamide selection (see WO 9830701) was
used, with additional modifications listed below. Agrobacterium was
grown to log phase in liquid minimal-A medium containing 100 .mu.M
acetosyringone and spectinomycin. Embryos were immersed in a log
phase suspension of Agrobacterium adjusted to obtain
3.times.10.sup.8 CFU's/ml. Embryos were then co-cultured on culture
medium with acetosyringone for 3 days at 20.degree. C. After 3 days
the embryos were returned to standard culture medium with 100 mg/l
carbinicillin added to kill residual Agrobacterium. After an
additional 4 days the segregating embryos were divided into GFP
positive and GFP negative populations and moved to fresh culture
medium with 50 mg/l cyanamide for selection. After 8 weeks the
numbers of transformed colonies were determined.
[0106] The results are summarized in the table below. Since the
PAT.about.GFP and LEC1 expression cassettes were co-segregating,
GFP expression was used to separate segregating transgenic
(PAT.about.GFP+/LEC1+) and non-transgenic (wild-type) embryos after
Agrobacterium-mediated transformation, and then these separate
populations were cultured and selected as independent groups. Using
embryos from three different ears co-segregating for GFP and LEC1,
the LEC1-containing embryos exhibited a much higher transformation
frequency demonstrating that ectopic LEC1 expression improves
re-transformation frequencies. Wild-type embryos (non-transgenic
segregants) from two ears did not produce transformants, while the
LEC1-containing embryos from the same ears produced
cyanimide-resistant transformants at approximately a 8.5%
frequency. In the third ear harvested and tested in this manner,
moCAH transformants were recovered at a 11.8% frequency for the
wild-type embryos, while for the LEC1 embryos from the same ear the
transformation frequency increased to 33.8%.
5TABLE V Tnx. Tnx. Frequency Frequency T0 transgenes # GFP+ # of
GFP- GFP+ GFP- Ear (co-segregating) embryos embryos embryos embryos
1 PHP16215 + 68 68 23/68 = 8/68 = GFP 33.8% 11.8% 2 PHP16102 + 47
70 4/47 = 0/70 GFP 8.5% 3 PHP16273 + 34 22 3/34 = 0/22 GFP 8.8%
[0107] Particle Gun Transformation Re-Transformations
[0108] As the starting point for particle gun-mediated
re-transformation experiments, regenerated Hi-II T0 transformants
were produced containing maize LEC1 expression cassettes and
UBI::moPAT.about.GFP::pinII. Transformants containing
UBI::moPAT.about.GFP::pinII and LEC1 expression cassettes were
tested; with LEC1 being driven by a nos promoter with additional
STOP codons added before the START in order to attenuate expression
(PHP16273), and a truncated version of the nos promoter containing
85 bases of the nos sequence (PHP16215). As a control, a
non-functional version of LEC1 was used, in which the LEC1 coding
sequence was frame-shifted by 1 position after the START codon,
resulting in essentially the same mRNA species but producing a
non-functional protein. Expression of this frame-shifted sequence
(abbreviated "f-shift" below) was driven by the In2 promoter
(PHP15636). As mentioned above for the functional LEC1 genes, this
f-shift LEC1 cassette co-segregated with GFP in the T1 progeny
embryos.
[0109] Transgenic Hi-II plants containing a co-segregating LEC1
expression cassette and the UBI::PAT.about.GFP expression cassette
were crossed to wild-type (non-transformed) Hi-II plants (using the
non-transformed parent as the pollen donor). As expected from such
a cross, the developing embryos on these ears segregated either for
transgene expression or wild-type. Embryos co-segregating for GFP
and LEC1 (functional and frame-shift versions) were transformed
using a particle gun using the standard Hi-II bombardment
transformation protocol (Songstad D. D. et al., In Vitro Cell Dev.
Biol. Plant 32:179-183, 1996). Cells were transformed by culturing
maize immature embryos (approximately 1-1.5 mm in length) onto 560P
medium containing N6 salts, Erikkson's vitamins, 0.69 g/l proline,
2 mg/l 2,4-D and 3% sucrose. After 4-5 days of incubation in the
dark at 28.degree. C., embryos were removed from 560P medium and
cultured, scutellum up, onto 560Y medium which is equivalent to
560P but contains 12% sucrose. Embryos were allowed to acclimate to
this medium for 3 h prior to transformation. The scutellar surface
of the immature embryos was targeted using particle bomardment with
a ubi:moCAH:pinII plasmid (PHP10675). Embryos were transformed
using the PDS-1000 Helium Gun from Bio-Rad at one shot per sample
using 650 PSI rupture disks. DNA delivered per shot averaged at
0.1667 ug. Following bombardment, all embryos were maintained on
560L medium (N6, salts, Eriksson's vitamins, 0.5 mg/l thiamine, 20
g/l sucrose, 1 mg/l 2,4-D, 2.88 g/l proline, 2.0 g/l gelrite, and
8.5 mg/l silver nitrate). After 2-7 days post-bombardment, all the
embryos from both treatments were transferred onto N6-based medium
containing 50 mg/l cyanamide (Pioneer 560P medium described above,
with 50 mg/l cyanamide). Plates were maintained at 28.degree. C. in
the dark and were observed for colony recovery with transfers to
fresh medium occurring every two to three weeks. Early in the
sub-culture regime, GFP+ and GFP- embryos were separated. These two
sub-populations were subsequently cultured and analyzed as separate
treatments. The PAT.about.GFP expression cassette and the LEC1
expression cassette co-segregate together, and thus the presence of
GFP expression is used to separate LEC1+ and LEC1- progeny for
analysis.
[0110] As seen in the table below, comparing PAT.about.GFP+/LEC1+
transgenic embryos with wild-type (non-transgenic) embryos from the
same ear showed that the overall recovery of cyanimide-resistant
transformants was much higher for the transgenic embryos. For the
first ear, a frame-shift control, there was no apparent
improvement. For the second and third ears, both expressing a
functional LEC1 protein, transformation frequencies increased from
0 (non-transformed) to 21.7% (transgenic) and from 9.1%
(non-transformed) to 88.5% (transformed), respectively.
6TABLE VI Tnx. Tnx. Frequency Frequency T0 transgenes # GFP+ # of
GFP- GFP+ GFP- Ear (co-segregating) embryos embryos embryos embryos
1 PHP15636 + 65 72 3/65 = 3/72 = GFP 4.6% 4.2% 2 PHP16273 + 23 29
5/23 = 0/29 GFP 21.7% 3 PHP16215 + 26 11 23/26 = 1/11 = GFP 88.5%
9.1%
* * * * *