U.S. patent application number 10/215146 was filed with the patent office on 2003-05-15 for methods of using viral replicase polynucleotides and polypeptides.
This patent application is currently assigned to Pioneer Hi-Bred International, Inc.. Invention is credited to Bailey, Matthew A., Burnett, Ronald, Dilkes, Brian R., Gordon-Kamm, William J., Gregory, Carolyn A., Hoerster, Goerge J., Larkins, Brian A., Lowe, Keith S., Woo, Young Min.
Application Number | 20030093831 10/215146 |
Document ID | / |
Family ID | 22975023 |
Filed Date | 2003-05-15 |
United States Patent
Application |
20030093831 |
Kind Code |
A1 |
Gordon-Kamm, William J. ; et
al. |
May 15, 2003 |
Methods of using viral replicase polynucleotides and
polypeptides
Abstract
The invention provides novel methods of using viral replicase
polypeptides. Included are methods for increasing transformation
frequencies, providing a positive growth advantage, and modulating
cell division.
Inventors: |
Gordon-Kamm, William J.;
(Urbandale, IA) ; Lowe, Keith S.; (Johnston,
IA) ; Bailey, Matthew A.; (Des Moines, IA) ;
Gregory, Carolyn A.; (Greenlawn, NY) ; Hoerster,
Goerge J.; (Des Moines, IA) ; Larkins, Brian A.;
(Tucson, AZ) ; Dilkes, Brian R.; (Tucson, AZ)
; Burnett, Ronald; (Bethlehem, PA) ; Woo, Young
Min; (Tucson, AZ) |
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: |
22975023 |
Appl. No.: |
10/215146 |
Filed: |
August 8, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10215146 |
Aug 8, 2002 |
|
|
|
09627107 |
Jul 27, 2000 |
|
|
|
6452070 |
|
|
|
|
09627107 |
Jul 27, 2000 |
|
|
|
09257131 |
Feb 25, 1999 |
|
|
|
6284947 |
|
|
|
|
Current U.S.
Class: |
800/280 ;
435/468 |
Current CPC
Class: |
C12N 15/8261 20130101;
C12N 15/8209 20130101; Y02A 40/146 20180101; C12N 15/8207 20130101;
C12N 15/8201 20130101 |
Class at
Publication: |
800/280 ;
435/468 |
International
Class: |
A01H 005/00; C12N
015/82 |
Claims
What is claimed is:
1. A method for increasing transformation frequencies in a target
plant cell comprising introducing into the target plant cell an
isolated plant geminivirus replicase polypeptide.
2. The method of claim 2 wherein the geminivirus replicase
polypeptide is wheat dwarf virus Replicase.
3. The method of claim 3 wherein the geminivirus replicase
polypeptide is RepA.
4. The method of claim 1 further comprising introducing into the
target plant cell a polynucleotide of interest.
5. The method of claim 4 wherein the plant cell is from a monocot
or a dicot plant.
6. The method of claim 5 wherein the plant cell is from corn,
soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice,
barley, potato, tomato, and millet.
7. A method for providing a positive growth advantage in a target
plant cell comprising introducing into the target plant cell an
isolated plant geminivirus replicase polypeptide.
8. The method of claim 7 wherein the geminivirus replicase
polypeptide is wheat dwarf virus Replicase.
9. The method of claim 8 wherein the geminivirus replicase
polypeptide is RepA.
10. The method of claim 7 wherein the plant cell is a monocot or a
dicot.
11. The method of claim 10 wherein the plant cell is from corn,
soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice,
barley, potato, tomato, and millet.
12. A method for modulating cell division of a target plant cell
capable of dividing, comprising introducing into the target plant
cell an isolated viral replicase polypeptide.
13. The method of claim 12 wherein the polypeptide is wheat dwarf
virus Replicase.
14. The method of claim 13 wherein the polypeptide is RepA.
15. The method of claim 12 wherein the plant cell is from a monocot
or a dicot plant.
16. The method of claim 15 wherein the plant cell is from corn,
soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice,
barley, potato, tomato, and millet.
Description
[0001] This application is a divisional of co-pending application
U.S. Se. No. 09/627,107 filed Jul. 27, 2000, which is a divisional
of co-pending U.S. Pat. No. 6,284,947 filed Feb. 25, 1999; the
disclosures of which are incorporated herein by reference.
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 varieties. 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 introduced into and evaluated directly in
commercial hybrids.
[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] In spite of increases in yield and harvested area worldwide,
it is predicted that over the next ten years, meeting the demand
for corn will require an additional 20% increase over current
production (Dowswell, C. R., Paliwal, R. L., Cantrell, R. P. 1996.
Maize in the Third World, Westview Press, Boulder, Colo.).
[0013] The components most often associated with maize productivity
are grain yield or whole-plant harvest for animal feed (in the
forms of silage, fodder, or stover). Thus the relative growth of
the vegetative or reproductive organs might be preferred, depending
on the ultimate use of the crop. Whether the whole plant or the ear
are harvested, overall yield will depend strongly on vigor and
growth rate. It would therefore be valuable to develop new methods
that contribute to the increase in crop yield.
SUMMARY OF THE INVENTION
[0014] It is an object of the present invention to provide methods
for modulating DNA replication in a transgenic plant.
[0015] It is another object of the present invention to provide a
method for increasing the number of cells undergoing cell
division.
[0016] It is another object of the present invention to provide a
method for increasing crop yield.
[0017] It is another object of the present invention to provide a
method for improving transformation frequencies.
[0018] It is another object of the present invention to provide a
method for improving transformation efficiency in cells from
various sources.
[0019] It is another object of the present invention to provide a
method for providing a positive growth advantage in a plant.
[0020] Therefore, in one aspect, the present invention provides a
method for increasing transformation frequencies comprising
introducing into a target cell a viral replicase polynucleotide
operably linked to a promoter driving expression in the target cell
or introducing a viral replicase polypeptide.
[0021] In another aspect the present invention provides a method
for increasing crop yield comprising introducing into a plant cell
an isolated viral replicase polynucleotide operably linked to a
promoter driving expression in the plant cell.
[0022] In another aspect the invention provides a method for
providing a positive growth advantage in a target cell comprising
introducing into the target cell an isolated viral replicase
polynucleotide operably linked to a promoter driving expression in
the target cell.
[0023] In another aspect the invention provides a method for
modulating cell division of target cells comprising introducing
into the target cell an isolated viral replicase polynucleotide in
sense or antisense orientation operably linked to a promoter
driving expression in the target cell or introducing an isolated
viral replicase polypeptide.
[0024] In another aspect the invention provides a method for
transiently modulating cell division of target cells comprising
introducing into the target cells an isolated viral replicase
polynucleotide in sense or antisense orientation operably linked to
a promoter driving expression in the target cells, an isolated
viral replicase polypeptide, or an antibody directed against a
viral replicase polypeptide.
[0025] In another aspect the invention provides a method for
providing a means of positive selection comprising (a) introducing
into a target cell an isolated viral replicase polynucleotide
operably linked to a promoter driving expression in the target cell
or an isolated viral replicase polypeptide and (b) selecting for
cells exhibiting positive growth advantage.
DETAILED DESCRIPTION OF THE INVENTION
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1: Comparison of transient GUS expression with and
without a Rep-expression cassette.
[0027] FIG. 2: Micrograph comparison of GFP fluorescence in cells
bombarded with and without a Rep-expression cassette.
[0028] FIG. 3: Comparison of cell cycle profile in callus
transformed with and without a Rep-expression cassette.
DEFINITIONS
[0029] The term "isolated" refers to material, such as a nucleic
acid or a protein, which is: (1) substantially or essentially free
from components which normally accompany or interact with the
material as found in its naturally occurring environment or (2) if
the material is in its natural environment, the material has been
altered by deliberate human intervention to a composition and/or
placed at a locus in the cell other than the locus native to the
material.
[0030] 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.
[0031] As used here, "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.
[0032] As used herein, "functional variant" or "functional
derivative" or "functional fragment" are used interchangeably. As
applied to polypeptides, the functional variant or derivative is a
fragment, a modified polypeptide, or a synthetic polypeptide that
stimulates DNA replication in a manner similar to the wild-type
gene products, Rep and RepA.
[0033] As used herein, "viral replicase polypeptides" refers to
polypeptides capable of stimulating DNA replication. The
polypeptides are intended to include functional variants,
fragments, and derivatives. The polypeptides exhibit the function
of binding to the family of retinoblastoma (Rb) proteins, or
Rb-associated proteins, or functional Rb homologs. The polypeptides
include functional variants or derivatives of viral replicase
proteins, and/or functional homologues. The polypeptides include
proteins encoded by genes in the viral genome that are commonly
referred to as "replication proteins", "replication associated
proteins", or "replication initiation proteins". The polypeptide
includes proteins from viruses in which all the "replication
associated" or "replication" functions are encoded as a single
protein, and those in which these functions are carried out by more
than one protein, irrespective of whether proper or "inappropriate"
splicing has occurred prior to translation (thus including both the
polypeptide encoded by the C1 Open Reading Frame, and the
polypeptide encoded by the C1-C2 fusion or properly spliced
C1-C2).
[0034] As used herein, "viral replicase polynucleotide" refers to
polynucleotides coding for a viral replicase polypeptide, including
functional variants, derivatives, fragments, or functional homologs
of characterized viral replicase polynucleotides.
[0035] As used herein, "plant" includes but is not limited to plant
cells, plant tissue and plant seeds.
[0036] The present invention provides novel methods of using viral
replicase polypeptides and polynucleotides. Included are methods
for increasing transformation frequencies, increasing crop yield,
providing a positive growth advantage, modulating cell division,
transiently modulating cell division, and for providing a means of
positive selection.
[0037] Viral replicase polynucleotides, functional variants and/or
functional homologs from any virus can be used in the methods of
the invention as long as the expressed polypeptides exhibit Rb
binding function, and/or stimulates DNA replication.
[0038] Examples of suitable plant viruses include wheat dwarf
virus, maize streak virus, tobacco yellow dwarf virus, tomato
golden mosaic virus, abutilon mosaic virus, cassava 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 cassava mosaic virus, and the bean yellow dwarf
virus.
[0039] Other viral proteins that bind Rb-related peptides include
the large-T antigen from SV40, adenovirus type 5 E1A protein, and
human papilloma virus type 16 - E7. Replicase from the wheat dwarf
virus has been sequenced and functionally characterized and is
therefore preferred. 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.
[0040] Viral replicase polynucleotides useful in the present
invention can be obtained using (a) standard recombinant methods,
(b) synthetic techniques, or combinations thereof.
[0041] Viral replicase polynucleotides and functional variants
useful in the invention can be obtained using primers that
selectively hybridize under stringent conditions. Primers are
generally at least 12 bases in length and can be as high as 200
bases, but will generally be from 15 to 75, preferably from 15 to
50. Functional fragments can be identified using a variety of
techniques such as restriction analysis, Southern analysis, primer
extension analysis, and DNA sequence analysis.
[0042] Variants of the nucleic acids can be obtained, for example,
by oligonucleotide-directed mutagenesis, linker-scanning
mutagenesis, mutagenesis using the polymerase chain reaction, and
the like. See, for example, Ausubel, pages 8.0.3-8.5.9. Also, see
generally, McPherson (ed.), DIRECTED MUTAGENESIS: A Practical
approach, (IRL Press, 1991). Thus, the present invention also
encompasses DNA molecules comprising nucleotide sequences that have
substantial sequence similarity with the inventive sequences.
Conservatively modified variants are preferred.
[0043] Nucleic acids produced by sequence shuffling of viral
replicase polynucleotides can also be used. Sequence shuffling is
described in PCT publication No. 96/19256. See also, Zhang, J.- H.,
et al. Proc. Natl. Acad. Sci. USA 94:4504-4509 (1997).
[0044] Also useful are 5' and/or 3' UTR regions for modulation of
translation of heterologous coding sequences. Positive sequence
motifs include translational initiation consensus sequences (Kozak,
Nucleic Acids Res.15:8125 (1987)) and the 7-methylguanosine cap
structure (Drummond et al., Nucleic Acids Res. 13:7375 (1985)).
Negative elements include stable intramolecular 5' UTR stem-loop
structures (Muesing et al., Cell 48:691 (1987)) and AUG sequences
or short reading frames 5' of the appropriate AUG in the 5' UTR
(Kozak, supra, Rao et al., Mol. and Cell. Biol. 8:284 (1988)).
[0045] Further, the polypeptide-encoding segments of the
polynucleotides can be modified to alter codon usage. Codon usage
in the coding regions of the polynucleotides of the present
invention can be analyzed statistically using commercially
available software packages such as "Codon Preference" available
from the University of Wisconsin Genetics Computer Group (see
Devereaux et al., Nucleic Acids Res. 12:387-395 (1984)) or
MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.).
[0046] For example, the polynucleotides can be optimized for
enhanced or suppressed expression in plants. See, for example,
EPA0359472; WO91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci.
USA 88:3324-3328; and Murray et al. (1989) Nucleic Acids Res.
17:477-498. In this manner, the genes can be synthesized utilizing
species-preferred codons. See, for example, Murray et al. (1989)
Nucleic Acids Res. 17:477-498, the disclosure of which is
incorporated herein by reference.
[0047] The nucleic acids may conveniently comprise a multi-cloning
site comprising one or more endonuclease restriction sites inserted
into the nucleic acid to aid in isolation of the polynucleotide.
Also, translatable sequences may be inserted to aid in the
isolation of the translated polynucleotide of the present
invention. For example, a hexa-histidine marker sequence provides a
convenient means to purify the proteins of the present
invention.
[0048] The polynucleotides can be attached to a vector, adapter,
promoter, transit peptide or linker for cloning and/or expression
of a polynucleotide of 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.).
[0049] To construct genomic libraries, large segments of genomic
DNA are generated by random fragmentation. Examples of appropriate
molecular biological techniques and instructions are found in
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,
Cold Spring Harbor Laboratory, Vols. 1-3 (1989), Methods in
Enzymology, Vol.152: Guide to Molecular Cloning Techniques, Berger
and Kimmel, Eds., San Diego: Academic Press, Inc. (1987), Current
Protocols in Molecular Biology, Ausubel et al., Eds., Greene
Publishing and Wiley-Interscience, New York (1995); Plant Molecular
Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin
(1997). Kits for construction of genomic libraries are also
commercially available.
[0050] The genomic library can be screened using a probe based upon
the sequence of a nucleic acid used in the present invention. Those
of skill in the art will appreciate that various degrees of
stringency of hybridization can be employed in the assay; and
either the hybridization or the wash medium can be stringent. The
degree of stringency can be controlled by temperature, ionic
strength, pH and the presence of a partially denaturing solvent
such as formamide.
[0051] Typically, stringent hybridization conditions will be those
in which the salt concentration is less than about 1.5 M Na ion,
typically about 0.01 to 1.0 M Na ion concentration (or other salts)
at pH 7.0 to 8.3 and the temperature is at least about 30.degree.
C. for short probes (e.g., 10 to 50 nucleotides) and at least about
60.degree. C. for long probes (e.g., greater than 50 nucleotides).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide.
[0052] Preferably the hybridization is conducted under low
stringency conditions which include hybridization with a buffer
solution of 30% formamide, 1 M NaCl, 1% SDS (sodium dodecyl
sulfate) at 37.degree. C., and a wash in 1.times. to 2.times. SSC
(20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50.degree. C.
More preferably the hybridization is conducted under moderate
stringency conditions which include hybridization in 40% formamide,
1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.5.times. to
1.times. SSC at 55.degree. C. Most preferably the hybridization is
conducted under high stringency conditions which include
hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C.,
and a wash in 0.1.times. SSC at 60.degree. C.
[0053] An extensive guide to the hybridization of nucleic acids is
found in Tijssen, Laboratory Techniques in Biochemistry and
Molecular Biology--Hybridization with Nucleic Acid Probes, Part I,
Chapter 2 "Overview of principles of hybridization and the strategy
of nucleic acid probe assays", Elsevier, New York (1993); and
Current Protocols in Molecular Biology, Chapter 2, Ausubel, et aL.,
Eds., Greene Publishing and Wiley-Interscience, New York (1995).
Often, cDNA libraries will be normalized to increase the
representation of relatively rare cDNAs.
[0054] The nucleic acids can be amplified from nucleic acid samples
using amplification techniques. For instance, polymerase chain
reaction (PCR) technology can be used to amplify the sequences of
polynucleotides of the present invention and related genes directly
from genomic DNA or libraries. PCR and other in vitro amplification
methods may also be useful, for example, to clone nucleic acid
sequences that code for proteins to be expressed, to make nucleic
acids to use as probes for detecting the presence of the desired
mRNA in samples, for nucleic acid sequencing, or for other
purposes.
[0055] Examples of techniques useful for in vitro amplification
methods are found in Berger, Sambrook, and Ausubel, as well as
Mullis et al., U.S. Pat. No. 4,683,202 (1987); and, PCR Protocols A
Guide to Methods and Applications, Innis et al., Eds., Academic
Press Inc., San Diego, Calif. (1990). Commercially available kits
for genomic PCR amplification are known in the art. See, e.g.,
Advantage-GC Genomic PCR Kit (Clontech). The T4 gene 32 protein
(Boehringer Mannheim) can be used to improve yield of long PCR
products.
[0056] PCR-based screening methods have also been described.
Wilfinger et al. describe a PCR-based method in which the longest
cDNA is identified in the first step so that incomplete clones can
be eliminated from study. BioTechniques, 22(3): 481-486 (1997).
[0057] The nucleic acids can also be prepared by direct chemical
synthesis by methods such as the phosphotriester method of Narang
et al., Meth. Enzymol. 68:90-99 (1979); the phosphodiester method
of Brown et al., Meth. Enzymol. 68:109-151 (1979); the
diethylphosphoramidite method of Beaucage et al., Tetra. Lett.
22:1859-1862 (1981); the solid phase phosphoramidite triester
method described by Beaucage and Caruthers, Tetra. Letts. 22(20):
1859-1862 (1981), e.g., using an automated synthesizer, e.g., as
described in Needham-VanDevanter et al., Nucleic Acids Res.,
12:6159-6168 (1984); and, the solid support method of U.S. Pat. No.
4,458,066.
[0058] Expression cassettes comprising the isolated viral replicase
nucleic acids are also provided. An expression cassette will
typically comprise a polynucleotide operably linked to
transcriptional initiation regulatory sequences that will direct
the transcription of the polynucleotide in the intended host cell,
such as tissues of a transformed plant.
[0059] 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, New York; (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.
[0060] For example, plant expression cassettes may include (1) a
viral replicase nucleic acid under the transcriptional control of
5' and 3' regulatory sequences and (2) a dominant selectable
marker. Such plant 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.
[0061] 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, the Smas promoter, the
cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439),
the Nos promoter, the pEmu promoter, the rubisco promoter, the
GRP1-8 promoter and other transcription initiation regions from
various plant genes known to those of skill.
[0062] Examples of inducible promoters are the Adh1 promoter which
is inducible by hypoxia or cold stress, the Hsp70 promoter which is
inducible by heat stress, and the PPDK promoter which is inducible
by light. Also useful are promoters which are chemically
inducible.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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).
[0067] The vector comprising the polynucleotide sequences useful in
the present invention will typically 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.
[0068] 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 s 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.
[0069] 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.
[0070] The viral replicase 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)
85:8805-809 (1988); and Hiaft et al., U.S. Pat. No. 4,801,340.
[0071] 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.
[0072] 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).
[0073] A variety of cross-linking agents, alkylating agents and
radical generating species as pendant groups on polynucleotides of
the present invention can be used to bind, label, detect, and/or
cleave nucleic acids. For example, Vlassov, V. V., et al., Nucleic
Acids Res (1986) 14:4065-4076, describe covalent bonding of a
single-stranded DNA fragment with alkylating derivatives of
nucleotides complementary to target sequences, report of similar
work by the same group is that by Knorre, D. G., et al., Biochimie
(1985) 67:785-789. Iverson and Dervan also showed sequence-specific
cleavage of single-stranded DNA mediated by incorporation of a
modified nucleotide which was capable of activating cleavage (J Am
Chem Soc (1987) 109:1241-1243). Meyer, R. B., et al., J Am Chem Soc
(1989) 111:8517-8519, effect covalent crosslinking to a target
nucleotide using an alkylating agent complementary to the
single-stranded target nucleotide sequence. A photoactivated
crosslinking to single-stranded oligonucleotides mediated by
psoralen was disclosed by Lee, B. L., et al., Biochemistry (1988)
27:3197-3203. Use of crosslinking in triple-helix forming probes
was also disclosed by Home et al., J Am Chem Soc (1990)
112:2435-2437. Use of N4, N4-ethanocytosine as an alkylating agent
to crosslink to single-stranded oligonucleotides has also been
described by Webb and Matteucci, J Am Chem Soc (1986)
108:2764-2765; Nucleic Acids Res (1986) 14:7661-7674; Feteritz et
al., J. Am. Chem. Soc. 113:4000 (1991). Various compounds to bind,
detect, label, and/or cleave nucleic acids are known in the art.
See, for example, U.S. Pat. Nos. 5,543,507; 5,672,593; 5,484,908;
5,256,648; and, 5,681,941.
[0074] Proteins useful in the present invention include proteins
derived from the native protein by deletion (so-called truncation),
addition or substitution of one or more amino acids at one or more
sites in the native protein. In constructing variants of the
proteins of interest, modifications will be made such that variants
continue to possess the desired activity.
[0075] For example, amino acid sequence variants of the polypeptide
can be prepared by mutations in the cloned DNA sequence encoding
the native protein of interest. Methods for mutagenesis and
nucleotide sequence alterations are well known in the art. See, for
example, Walker and Gaastra, eds. (1983) Techniques in Molecular
Biology (MacMillan Publishing Company, New York); Kunkel (1985)
Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods
Enzymol. 154:367-382; Sambrook et al. (1989) Molecular Cloning: A
Laboratory Manual (Cold Spring Harbor, New York); U.S. Pat. No.
4,873,192; and the references cited therein; herein incorporated by
reference. Guidance as to appropriate amino acid substitutions that
do not affect biological activity of the protein of interest may be
found in the model of Dayhoff et al. (1978) Atlas of Protein
Sequence and Structure (Natl. Biomed. Res. Found., Washington,
D.C.), herein incorporated by reference. Conservative
substitutions, such as exchanging one amino acid with another
having similar properties, may be preferred.
[0076] The present invention includes catalytically active
polypeptides (i.e., enzymes). Catalytically active polypeptides
will generally have a specific activity of at least 20%, 30%, or
40%, and preferably at least 50%, 60%, or 70%, and most preferably
at least 80%, 90%, or 95% that of the native (non-synthetic),
endogenous polypeptide. Further, the substrate specificity
(k.sub.cat/K.sub.m) is optionally substantially similar to the
native (non-synthetic), endogenous polypeptide. Typically, the
K.sub.m will be at least 30%, 40%, or 50%, that of the native
(non-synthetic), endogenous polypeptide; and more preferably at
least 60%, 70%, 80%, or 90%. Methods of assaying and quantifying
measures of enzymatic activity and substrate specificity
(k.sub.cat/K.sub.m), are well known to those of skill in the
art.
[0077] The methods of the present invention can be used with any
cell such as bacteria, yeast, insect, mammalian, or preferably
plant cells. The transformed cells produce viral replicase
protein.
[0078] 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.
[0079] 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.
[0080] 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)).
[0081] In some aspects of the invention, viral replicase proteins
are introduced into a cell to increase cell division. Synthesis of
heterologous proteins in yeast is well known. See Sherman, F., et
al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory
(1982). Two widely utilized yeast for production of eukaryotic
proteins are Saccharomyces cerevisiae and Pichia pastors. Vectors,
strains, and protocols for expression in Saccharomyces and Pichia
are known in the art and available from commercial suppliers (e.g.,
Invitrogen). Suitable vectors usually have expression control
sequences, such as promoters, including 3-phosphoglycerate kinase
or alcohol oxidase, and an origin of replication, termination
sequences and the like as desired.
[0082] The protein can be isolated from yeast by lysing the cells
and applying standard protein isolation techniques to the lysates.
The monitoring of the purification process can be accomplished by
using Western blot techniques or radioimmunoassay of other standard
immunoassay techniques.
[0083] The proteins useful in the present invention can also be
constructed using non-cellular synthetic methods. Techniques for
solid phase synthesis are described by Barany and Merrifield,
Solid-Phase Peptide Synthesis, pp. 3-284 in The Peptides: Analysis,
Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis,
Part A.; Merrifield et al., J. Am. Chem. Soc. 85: 2149-2156 (1963),
and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce
Chem. Co., Rockford, Ill. (1984). Proteins of greater length may be
synthesized by condensation of the amino and carboxy termini of
shorter fragments. Methods of forming peptide bonds by activation
of a carboxy terminal end (e.g., by the use of the coupling reagent
N,N'-dicycylohexylcarbodiimide) are known to those of skill.
[0084] The proteins useful in this invention may be purified to
substantial purity by standard techniques well known in the art,
including detergent solubilization, selective precipitation with
such substances as ammonium sulfate, column chromatography,
immunopurification methods, and others. See, for instance, R.
Scopes, Protein Purification: Principles and Practice,
Springer-Verlag: New York (1982); Deutscher, Guide to Protein
Purification, Academic Press (1990). For example, antibodies may be
raised to the proteins as described herein. Purification from E.
coli can be achieved following procedures described in U.S. Pat.
No. 4,511,503. Detection of the expressed protein is achieved by
methods known in the art, for example, radioimmunoassays, Western
blotting techniques or immunoprecipitation.
[0085] Expressing viral replicase polypeptides is expected to
provide a positive growth advantage and increase crop yield.
[0086] In a preferred embodiment, the invention can be practiced in
a wide range of plants such as monocots and dicots. In a especially
preferred embodiment, the methods of the present invention are
employed in corn, soybean, sunflower, safflower, potato, tomato,
sorghum, canola, wheat, alfalfa, cotton, rice, barley and
millet.
[0087] The method of transformation/transfection is not critical to
the invention; various methods of transformation or transfection
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 to effect phenotypic changes in
the organism. Thus, any method that provides for efficient
transformation/transfection may be employed.
[0088] 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 plant. Isolated nucleic acid acids
of 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.
[0089] 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.
[0090] 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). 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.
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. See, U.S. Pat. No. 5,591,616.
[0091] 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).
[0092] 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.
5,550,318.
[0093] 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).
[0094] 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).
[0095] Animal and lower eukaryotic (e.g., yeast) host cells are
competent or rendered competent for transfection 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).
[0096] 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).
[0097] Plants cells transformed with a plant expression vector 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).
[0098] 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.
[0099] 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, .sub.3rd edition, Sprague and
Dudley Eds., American Society of Agronomy, Madison, Wis.
(1988).
[0100] 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.
[0101] 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.
[0102] Parts obtained from the regenerated plant, such as flowers,
seeds, leaves, branches, fruit, and the like are included in the
invention, provided that these parts comprise cells comprising the
isolated viral replicase nucleic acid. 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.
[0103] Transgenic plants expressing a selectable marker can be
screened for transmission of the viral replicase nucleic acid, 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. The RNA-positive plants can then
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.
[0104] Plants that can be used in the method of the invention vary
broadly and include monocotyledonous and dicotyledonous plants.
Preferred plants include corn, soybean, sunflower, sorghum, canola,
wheat, alfalfa, cofton, rice, barley, potato, tomato, and
millet.
[0105] Seeds derived from plants regenerated from transformed plant
cells, plant parts or plant tissues, or progeny derived from the
regenerated transformed plants, may be used directly as feed or
food, or further processing may occur.
[0106] Expression of the viral replicase nucleic acids in plants,
such as maize, is expected to enhance growth and biomass
accumulation. Other more specialized applications exist for these
nucleic acids at the whole plant level.
[0107] The present invention will be further described by reference
to the following detailed examples.
[0108] 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. All
publications, patents, and patent applications cited herein are
hereby incorporated by reference.
EXAMPLES
Example 1
Replicase Constructs
[0109] The replicase gene was obtained from Joachim Messing in the
vector pWI-1 1, and was re-designated P100. Using P100 as the
source, the replicase structural gene was cloned into an
intermediate vector containing the 35S promoter and a 35S 3'
sequence (for expression studies in dicotyledonous species, such as
tobacco; designated P101 made in the Larkins Lab, Univ. of
Arizona). From this intermediate plasmid, the RepA structural gene
and the 35S 3' sequence were excised using the restriction enzyme
NcoI and PstI, and cloned into P101 (gamma zein
promoter::uidA::Gamma zein 3' region; after the removal of the GUS
structural gene from P101 using NcoI/PstI). This resulted in a
final construct containing an expression cassette with a maize
gamma zein promoter sequence (GZ), the RepA coding sequence, a 35S
terminator and a gamma zein 3' sequence (GZ'). Thus, the expression
cassette had the configuration GZ:: RepA::35S::GZ'P102.
[0110] A derivative of the pWI-11 vector, with both iudA (encoding
GUS expression) and rep gene expression being driven by the
bi-directional promoter elements in the WDV long intergenic region
(WDV-LIR) was also provided by the Messing lab (pWI-GUS).
Example 2
Replicase results in increased transient expression of co-delivered
transgenes
[0111] The plasmids listed in Table I below were used to evaluate
the influence of Rep on transient expression of co-delivered
transgenes. The SuperMAS promoter is that described by Ni et al.,
1996, Sequence-specific interactions of wound-inducible nuclear
factors with mannopine synthase 2' promoter wound responsive
elements, Plant Mol. Biol. 30:77-96. The visible marker genes, GUS
(b-glucoronidase; Jefferson R. A., Plant Mol. Biol. Rep. 5:387,
1987) and GFP (green fluorescent protein; Chalfie et al., Science
263:802, 1994) have been described, as has the maize-optimized GFP
(GFPm; see copending U.S. patent application WO 97/41228). The
Ubiquitin promoter has been described (Christensen et al., Plant
Mol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol.
Biol. 18:675-689 (1992), as have the pinII (An et al., 1989, Plant
Cell 1:115-122) and 35S (Odell et al., 1985, Nature 313:810-812) 3'
regions used in these expression cassettes.
1TABLE I Constructs used to evaluate the effect of replicase
expression on transient expression of co-delivered transgenes.
Plasmid Description P103 SuperMAS::GUS::pinll 3' region P104
UBI::moPAT::CaMV35S 3' region P105 UBI::GFPm::pinll P100 WDV-LIR
promoter::replicase
[0112] GFP expression in Maize
[0113] Transformation of the Rep plasmid DNA, P100, into the
Pioneer Hi-Bred Int'l. Inc. proprietary inbred, N38, 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 the 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 with either a UBI::GFPm::pinII plasmid+a
UBI::maize-optimized PAT::pinII plasmid (P105,control treatment) or
with a combination of the UBI::GFPm::pinII plasmid P104+ the
replicase plasmidP100. Embryos were transformed using the PDS-1000
Helium Gun from Bio-Rad at one shot per sample using 650PSI rupture
disks. DNA delivered per shot averaged at 0.0667 ug. An equal
number of embryos per ear were bombarded with either the control
DNA mixture or the Rep/GFP DNA mixture. 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 evaluated for transient GFP expression.
[0114] In both experiments, greater numbers of cells on the
scutellar surface transiently expressed GFP in the
replicase-containing treatment. In experiment #1 with genotype N38,
a mean of only 12 cells per embryo transiently expressed GFP in the
treatment without replicase, while in the replicase-treated embryos
the mean number of GFP-expressing cells was almost 20-fold greater
(see Table II below). In the second experiment (Table III below),
transient GFP expression in the replicase-containing treatments was
approximately 6.5-fold greater than in the control treatments (no
replicase).
2TABLE II Maize Experiment #1: Transient GFP expression is
stimulated by Replicase Genotype & Treatment GFP-expressing
Explant (plasmids used) cells/embryo* Mean N38 immature P104, P100
165, 290, 233 embryos 413, 149, 148 N38 immature P104, P105 1, 22,
13 12 embryos
[0115]
3TABLE III Maize Experiment #2: Transient GFP expression Is
stimulated by Replicase Genotype & Treatment GFP-expressing
Explant (plasmids used) cells/embryo* Mean N38 immature P104, P100
1122, 108, 285, 358 embryos (insert) 27, 249 N38 immature P104,
P105 240, 10, 11, 0, 0 52 embryos *the number of GFP-expressing
cells per embryo was averaged across all 25 embryos on the
plate.
[0116] Soybean
[0117] Tissue was excised from coyledons and placed on MS-based
medium. A mixture of plasmid DNA, containing equal amounts of a
SuperMas::GUS::pinII plasmid (P103) and the WDV-LIR::replicase
plasmid (P100) was delivered into cells on the surface of the
colyledon explants using particle-mediated delivery similar to that
descibed for maize above. As a control, SuperMas::GUS::pinII
plasmid (P103)+UBI::moPAT::CaMV- 35S (P105) was introduced into the
same target cells using an equal number of cotyledonary tissue
pieces.
[0118] In the replicase-treatment, greater numbers of transiently
expressing cells were observed on the cotyledon after GUS staining
(see FIG. 1). In addition, for cells exhibiting transient gene
expression, the level of expression as judged by relative intensity
of histochemical staining appeared greater in replicase-treated
tissues (as compared to controls).
Example 3
RepA increases growth rates in early-developing stable maize
transformants
[0119] Transformation of the RepA plasmid DNA (P102), P102in Hi-II
followed 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 either a
UBI::moPAT-GFPm::pinII plasmid (P106 alone as a control treatment)
or with a combination of the UBI::moPAT-GFPm::pinII plasmid
(P106)+the GZ::RepA::35S:GZ' plasmid (P102). Embryos were
transformed using the PDS-1000 Helium Gun from Bio-Rad at one shot
per sample using 650PSI rupture disks. DNA delivered per shot
averaged at 0.0667 ug. An equal number of embryos per ear were
bombarded with either the control DNA (PAT-GFP) or the RepA/PAT-GFP
DNA mixture. 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 3 mg/l bialaphos Pioneer 560P medium described above,
with no proline and with 3 mg/l bialaphos). Plates were maintained
at 28.degree. C. in the dark and were observed for colony recovery
with transfers to fresh medium occurring every two weeks. Two weeks
after DNA delivery, the newly-forming callus was examined using
epifluorescence under the dissecting microscope (using
commercially-available filter combinations for GFP excitation and
emission).
[0120] At 2 weeks post-bombardment, numerous cells on the surface
of the scutellar-derived tissue were expressing GFP in the control
treatment (no RepA), but all expressing foci consisted of single
cell. No multicellular GFP-expressing clusters were observed in the
control. At this same time-point, 2-weeks after DNA-delivery, the
same sprinkling of single-celled GFP-expressing foci were observed
on the surface of the tissue that had received the RepA/PAT-GFP
mixture. However, numerous macroscopic GFP-expressing multicellular
clusters were also apparent (see FIG. 2). Many embryos were
observed with multiple transgenic microcalli developing on the
surface, with as many as 7 apparently- independent transformants
beginning to grow from a single embryo (this has never been
reported before for particle bombardment of maize).
[0121] After 3 weeks, GFP-expressing single cells could still be
observed in both treatments, although the frequency had declined.
In the control treatment, a solitary GFP-expressing multicellular
colony we observed to be developing on one embryo (out of 50
total). In the RepA-treated embryos, the growth rate of the
developing transgenic calli continued to be very rapid. Many of the
multiple colonies apparently growing from single embryos were
already in danger of co-mingling by growing together into a single
mass. Many colonies were picked off the embryos to grow them
separately. At 5 weeks post-bombardment, many RepA colonies
continued to grow rapidly (some may have been too small to survive
independently). While growing rapidly, these RepA-treated
transgenic calli maintain a healthy embryogenic character.
Example 4
RepA increases cell division rates in tobacco suspension culture
cells
[0122] For tobacco BY-2 suspension culture cells, the following
construct was used; 35S promoter::RepA::35S 3' region (P101).
Suspension cells were grown in a medium comprised of Murashige and
Skoog salts (Life Technologies, Inc., Grand Island, N.Y.), 100 mg/l
inositol, 1 mg/l thiamine, 180 mg/l KH2PO4, 30 g/l sucrose, and 2
mg/l 2,4-D, subcultured every 7-10 days, and grown on a gyratory
shaker at 150 RPM, 24.degree. C. in the dark. Three days after
subculturing, cells were pipetted onto solidified agar medium for
bombardment, and left in the dark for 24 hours. Bombardment was
performed using a BioRad PDS-1000, using helium at 650 PSI and 25
inches Hg, with 8 cm distance between the stopping plate and petri
dish. All cells were shot once with 500 ng gold and 0.5 .mu.g DNA.
All the treated cells received a plasmid containing a 35S::GFP::35S
expression cassette (P108), with half receiving an additional
plasmid containing the 35S::RepA::35S cassette. After bombardment,
the cells were monitored for GFP expression and cell division.
[0123] After 24 hours, GFP-expressing cells were scored as
non-dividing (single fluorescent cells) or as having divided during
the intervening 24-hour period (i.e. GFP-expressing doublets with
the characteristic newly-formed division plate between the two
fluorescent daughter cells). For the control treatment (GFP alone),
37.5% (with a standard error of 1.8, calculated for three
replicates) of the total number of GFP-expressing cells had
undergone division during this period. In the treatment where
GFP+RepA expression cassettes were introduced simultaneously, the
percentage of GFP-expressing cells that had undergone division
increased substantially to 45.7 (SE=5.7).
Example 5
RepA increases maize transformation frequency
[0124] For transformation experiments, a construct was used in
which the RepA coding sequence was cloned into a maize expression
cassette (P102, described above). Delivery of the RepA gene in an
appropriate plant expression cassette (for example, in a
GZ::RepA::35S:GZ-containing plasmid) along with marker gene
cassettes was accomplished using particle bombardment. DNA was
introduced into maize cells capable of growth on suitable maize
culture medium (freshly isolated immature embryos). See Table IV
below for treatments. Immature embryos of the Hi-II genotype were
used as the target for co-delivery of plasmids. To assess the
effect on transgene integration, growth of bialaphos-resistant
colonies on selective medium was a reliable assay. Within 1-7 days
after DNA introduction, the embryos were moved onto culture medium
containing 3 mg/l of the selective agent bialaphos. Embryos, and
later callus, were transferred to fresh selection plates every 2
weeks. Four-six weeks after bombardment, bialaphos-resistant calli
were scored and transferred to separate plates to prevent mixing of
transformants as they continue to grow. Expression of the visible
scorable marker ( GUS or GFP) was used to confirm transformation.
In the RepA-treated embryos, higher numbers of stable transformants
were recovered (likely a direct result of increased integration
frequencies).
4TABLE IV Experimental design for assessing the influence of RepA
expression on recovery of stable maize transformants. Experiment #
Control Treatment 1 & 2 None included in E35S::bar::pinll +
UBI::GUS::pinll + experiment GZ::RepA::35S:GZ' 3
UBI::PATm-GFPm::pinll UBI::PATm-GFPm::pinll + GZ::RepA::35S:GZ'
[0125] Experiment #1. This experiment was originally designed to
test RepA expression in endosperm. Thus, we used all of the embryos
from the available Hi-II ears on this day to introduce RepA along
with the marker genes (P107 the construct containing Enhanced-35S
promoter::bar::pinII and UBI::GUS::pinII). The frequencies for
Hi-II transformation using P107 alone
(E35S::bar::pinII+UBI::GUS::pinII) during this period were
averaging between 2-3%, providing a good basis of comparison. In
this experiment, our transformation frequency with P107+GZ::RepA
(P102) was 8.8% (33 transformants /375 starting embryos).
[0126] Experiment #2. Again, the original intent of this experiment
was to generate endosperm-expressing RepA transformants (not to
compare transformation frequencies). As in the first experiment,
the observed result was unexpected; transformation frequency using
P107+GZ::RepA (P102) was 29.2% (73 transformants /250 starting
embryos). This represented approximately a 10-fold increase over
the 2-3% transformation frequencies observed in other experiments
conducted during this period using similar marker genes (the bar
gene to confer bialaphos resistance and GUS as a visible
marker).
[0127] Experiment #3. In this experiment, numerous ears were used.
Immature embryos were isolated from each ear, randomized on plates
and then split between each of the two treatments (+/-RepA). This
comparison used a total of 725 embryos per treatment, harvested
from a total of 29 ears (25 embryos/ear/treatment). Transformation
frequencies were calculated on a per-ear basis and then expressed
as the mean.
5 Mean Transformation Standard Treatment Frequency (%) Deviation
UBI::PATm-GFPm::pinll (Control) 2.2 1.8 UBI::PATm-GFPm::pinll +
17.0 8.5 GZ::RepA::35S:GZ'
[0128] This tightly controlled experiment validated the preliminary
results in Experiments #1 & #2. Across many replicates
(individual ears harvested on separate dates), the mean frequency
for RepA-treated immature embryos was over 7.5-fold greater than
for embryos treated solely with the control plasmid. For
particle-mediated transformation of Hi-II immature embryos, this is
a remarkable improvement in transformation frequency. The calli
recovered from the RepA treatments grew vigorously, were
embryogenic, and easily regenerated into plants. Plants regenerated
to date have appeared phenotypically normal, were both male and
female fertile, and transmitted the transgenes (and their
expression) to progeny in expected Mendelian ratios.
Example 6
RepA alters the cell cycle phenotype in cell populations from
transgenic calli
[0129] Transformation of Hi-II immature embryos was performed using
the prottocol described in Example 3. A mixture of plasmid DNA,
containing equal amounts of a E35S::bar::pinII+UBI::GUS::pinII
plasmid (P107) and a GZ::RepA35S::GZ' plasmid (P102), was delivered
into scutellar cells of the immature embryos using
particle-mediated delivery. As a control,
E35S::bar::pinII+UBI::GUS::pinII (P107) plasmid alone was
introduced into the same target cells on the surface of the
scutellum for an equal number of embryos. One week after particle
bombardment, all the embryos from both treatments were transferred
onto N6-based medium containing 3 mg/l bialaphos. After 6 weeks,
stable transformants were scored, and expression of a second marker
gene (GUS) was used to confirm the transgenic nature of the callus.
Transgenic callus expressing bar and GUS alone (from the control
treatment), or transgenic callus expressing bar, GUS and RepA were
used to isolate nuclei. For extraction of nuclei, callus was
macerated with a straight-edge razor blade in a buffer consisting
of 45 mM CgCL.sub.2, 30 mM sodium citrate, 20 mM MOPS buffer, 0.1%
v/v Triton X 100. For each callus event sampled, tissue
(approximately 1 cm.sup.3) was transferred to a Petri dish, and
macerated with a small volume of the chopping buffer. The resulting
suspension was then passed sequentially through 60 um and 20 um
sieves and transferred to a 15 ml centrifuge tube on ice. Tubes
were centrifuged at 100 g for 5 minutes at 4.degree. C. The
supernatant was decanted, the pellets resuspended in .about.750
.mu.l of staining solution (100 .mu.g/ml propidium iodide in
chopping buffer) and transferred to tubes for analysis in the flow
cytometer. Stained nuclei were analyzed on an EPICS-XL-MCL flow
cytometer using a 488 nm argon laser for excitation and measuring
emission from 500-550 nm. Collecting propidium iodide fluorescence
measurements on a per-nucleus basis (equivalent to the DNA content
per nucleus) permitted the assessment of cell cycle stages in the
callus-cell population.
[0130] The cell cycle profile from the control callus was typical
of maize callus cell populations, with a predominant G1 peak
(approximately 80%), a low percentage of S phase (8%), and a low
percentage of G2 (approximately 12%). In a RepA-treated callus
transformant, the cell cycle profile was dramatically shifted, with
approximately 7% G1, 8% S phase and 85% in the G2 phase (see FIG.
3).
Example 7
Transient RepA activity enhances transformation frequency
[0131] For this specific application (using transient RepA-mediated
cell cycle stimulation to increase transient integration
frequencies), it may be desirable to reduce the likelihood of
ectopic stable expression of the RepA gene. Strategies for
transient-only expression can be used. This includes delivery of
RNA (transcribed from the RepA gene), chemically end-modified DNA
expression cassettes that typically will not integrate, or RepA
protein along with the transgene cassettes to be integrated to
enhance transgene integration by transient stimulation of cell
division. Using well-established methods to produce RepA-RNA, this
can then be purified and introduced into maize cells using physical
methods such as microinjection, bombardment, electroporation or
silica fiber methods. For protein delivery, the gene is first
expressed in a bacterial or baculoviral system, the protein
purified and then introduced into maize cells using physical
methods such as microinjection, bombardment, electroporation or
silica fiber methods. Alternatively, RepA proteins are delivered
from Agrobacterium tumefaciens into plant cells in the form of
fusions to Agrobacterium virulence proteins. Fusions are
constructed between RepA and bacterial virulence proteins such as
VirE2, VirD2, or VirF which are known to be delivered directly into
plant cells. Fusions are constructed to retain both those
properties of bacterial virulence proteins required to mediate
delivery into plant cells and the RepA activity required for
enhancing transgene integration. This method ensures a high
frequency of simultaneous co-delivery of T-DNA and functional RepA
protein into the same host cell. The methods above represent
various means of using the RepA gene or its encoded product to
transiently stimulate DNA replication and cell division, which in
turn enhances transgene integration by providing an improved
cellular/molecular environment for this event to occur.
Example 8
Altering RepA expression stimulates the cell cycle and growth
[0132] Based on our observations, expression of RepA genes
increases cell division rates. Increases in division rate are
assessed in a number of different manners, being reflected in
smaller cell size, more rapid incorporation of radiolabeled
nucleotides, and faster growth (i.e. more biomass accumulation).
Delivery of the RepA in an appropriate plant expression cassette is
accomplished through numerous well- established methods for plant
cells, including for example particle bombardment, sonication, PEG
treatment or electroporation of protoplasts, electroporation of
intact tissue, silica-fiber methods, microinjection or
Agrobacterium-mediated transformation. The result of RepA gene
expression will be to stimulate the G1/S transition and hence cell
division, providing the optimal cellular environment for
integration of introduced genes (as per Example 1). This will
trigger a tissue culture response (cell divisions) in genotypes
that typically do not respond to conventional culture techniques,
or stimulate growth of transgenic tissue beyond the normal rates
observed in wild-type (non-transgenic) tissues. To demonstrate
this, the RepA gene is cloned into a cassette with a constitutive
promoter (i.e. either a strong maize promoter such as the ubiquitin
promoter including the first ubiquitin intron, or a weak
constitutive promoter such as nos). Either particle-mediated DNA
delivery or Agrobacterium-mediated delivery are used to introduce
the GZ::RepA::35S:GZ-containing plasmid along with a
UBI::bar:pinII-containin- g plasmid into maize cells capable of
growth on suitable maize culture medium. Such competent cells can
be from maize suspension culture, callus culture on solid medium,
freshly isolated immature embryos or meristem cells. Immature
embryos of the Hi-II genotype are used as the target for
co-delivery of these two plasmids, and within 1-7 days the embryos
are moved onto culture medium containing 3 mg/l of the selective
agent bialaphos. Embryos, and later callus, are transferred to
fresh selection plates every 2 weeks. After 6-8 weeks, transformed
calli are recovered. In treatments where both the bar gene and RepA
gene have been transformed into immature embryos, a higher number
of growing calli are recovered on the selective medium and callus
growth is stimulated (relative to treatments with the bar gene
alone). When the RepA gene is introduced without any additional
selective marker, transgenic calli can be identified by their
ability to grow more rapidly than surrounding wild-type
(non-transformed) tissues. Transgenic callus can be verified using
PCR and Southern analysis. Northern analysis can also be used to
verify which calli are expressing the bar gene, and which are
expressing the maize RepA gene at levels above normal wild-type
cells (based on hybridization of probes to freshly isolated mRNA
population from the cells).
[0133] Inducible Expression
[0134] The RepA gene can also be cloned into a cassette with an
inducible promoter such as the benzenesulfonamide-inducible
promoter. The expression vector is co-introduced into plant cells
and after selection on bialaphos, the transformed cells are exposed
to the safener (inducer). This chemical induction of RepA
expression results in stimulated G1/S transition and more rapid
cell division. The cells are screened for the presence of RepA RNA
by northern, or RT-PCR (using transgene specific probes/oligo
pairs), for RepA-encoded protein using RepA-specific antibodies in
Westerns or using hybridization. Increased DNA replication is
detected using BrdU labeling followed by antibody detection of
cells that incorporated this thymidine analogue. Likewise, other
cell cycle division assays could be employed, as described
above.
Example 9
Control of RepA gene expression using tissue-specific or
cell-specific promoters provides a differential growth
advantage
[0135] RepA gene expression using tissue-specific or cell-specific
promoters stimulates cell cycle progression in the expressing
tissues or cells. For example, using a seed-specific promoter will
stimulate cell division rate and result in increased seed biomass.
Alternatively, driving RepA expression with an tassel-specific
promoter will enhance development of this entire reproductive
structure.
[0136] Expression of RepA genes in other cell types and/or at
different stages of development will similarly stimulate cell
division rates. Similar to results observed in Arabidopsis (Doerner
et al., 1996), root-specific or root-preferred expression of RepA
will result in larger roots and faster growth (i.e. more biomass
accumulation).
Example 10
Meristem Transformation
[0137] Meristem transformation protocols rely on the transformation
of apical initials or cells that can become apicial initials
following reorganization due to injury or selective pressure. The
progenitors of these apical initials differentiate to form the
tissues and organs of the mature plant (i.e. leaves, stems, ears,
tassels, etc.). The meristems of most angiosperms are layered with
each layer having its own set of initials. Normally in the shoot
apex these layers rarely mix. In maize the outer layer of the
apical meristem, the L1, differentiates to form the epidermis while
descendents of cells in the inner layer, the L2, give rise to
internal plant parts including the gametes. The initials in each of
these layers are defined solely by position and can be replaced by
adjacent cells if they are killed or compromised. Meristem
transformation frequently targets a subset of the population of
apical initials and the resulting plants are chimeric. If for
example, 1 of 4 initials in the L1 layer of the meristem are
transformed only 1/4 of epidermis would be transformed. Selective
pressure can be used to enlarge sectors but this selection must be
non-lethal since large groups of cells are required for meristem
function and survival. Transformation of an apical initial with a
RepA expression cassette under the expression of a promoter active
in the apical meristem (either meristem specific or constitutive)
would allow the transformed cells to grow faster and displace
wildtype initials driving the meristem towards homogeneity and
minimizing the chimeric nature of the plant body. To demonstrate
this, the RepA gene is cloned into a cassette with a promoter that
is active within the meristem (i.e. a promoter active in
meristematic cells such as the maize histone, cdc2 or actin
promoter). Coleoptilar stage embryos are isolated and plated
meristem up on a high sucrose maturation medium (see Lowe et al.,
1997). The RepA expression cassette along with a reporter construct
such as Ubi:GUS:pinII can then be co-delivered (preferably 24 hours
after isolation) into the exposed apical dome using conventional
particle gun transformation protocols. As a control the RepA
construct can be replaced with an equivalent amount of pUC plasmid
DNA. After a week to 10 days of culture on maturation medium the
embryos can be transferred to a low sucrose hormone-free
germination medium. Leaves from developing plants can be sacrificed
for GUS staining. Transient expression of the RepA gene in meristem
cells, through stimulation of the G1.fwdarw.S transition, will
result in greater integration frequencies and hence more numerous
transgenic sectors. Integration and expression of the RepA gene
will impart a competitive advantage to expressing cells resulting
in a progressive enlargement of the transgenic sector. Due to the
enhanced growth rate in RepA-expressing meristem cells, they will
supplant wild-type meristem cells as the plant continues to grow.
The result will be both enlargement of transgenic sectors within a
given cell layer (i.e. periclinal expansion) and into adjacent cell
layers (i.e. anticlinal invasions). As an increasingly large
proportion of the meristem is occupied by RepA-expressing cells,
the frequency of RepA germlne inheritance goes up accordingly.
Example 11
Use of Flp/Frt system to excise the RepA cassette
[0138] In cases where the RepA gene has been integrated and RepA
expression is useful in the recovery of maize trangenics, but is
ultimately not desired in the final product, the RepA expression
cassette (or any portion thereof that is flanked by appropriate FRT
recombination sequences) can be excised using FLP-mediated
recombination (see co-pending U.S. Patent Application US
98/24640).
* * * * *