U.S. patent application number 09/907411 was filed with the patent office on 2003-03-06 for methods of transforming plants and identifying parental origin of a chromosome in those plants.
Invention is credited to Cole, Glenn S., Marsh, Wallace A., Meyer, Terry EuClaire, Mullen, Jeffrey A., Ranch, Jerome P., Scelonge, Christopher J., Soper, John F., Tomes, Dwight T., Wang, Jimei, Zhao, Zuo-Yu, Zhong, Gan-Yuan.
Application Number | 20030046724 09/907411 |
Document ID | / |
Family ID | 22816920 |
Filed Date | 2003-03-06 |
United States Patent
Application |
20030046724 |
Kind Code |
A1 |
Ranch, Jerome P. ; et
al. |
March 6, 2003 |
Methods of transforming plants and identifying parental origin of a
chromosome in those plants
Abstract
Methods for plant transformation, for improving transformation
efficiency, and for producing transgenic plants are provided. The
methods comprise crossing a recipient plant from a genetic line of
a plant species of interest with a donor plant selected from a
transformation competent genetic line of the same plant species or
of another closely related plant species to obtain a hybrid plant.
Tissues obtained from the hybrid plant are transformation
competent. These tissues can then be transformed with one or more
nucleotide sequences of interest and selected for transgenic events
having the nucleotide sequence of interest integrated within a
chromosome derived from the recipient plant. Transformed cells can
be selected and transgenic hybrid plants regenerated. The
nucleotide sequence of interest can be introgressed into the
genetic line from which the original recipient parent was derived,
or into other genetic lines. Transformed plants and seeds are
additionally provided.
Inventors: |
Ranch, Jerome P.; (West Des
Moines, IA) ; Marsh, Wallace A.; (Ankeny, IA)
; Meyer, Terry EuClaire; (Urbandale, IA) ; Tomes,
Dwight T.; (Van Meter, IA) ; Zhao, Zuo-Yu;
(Johnston, IA) ; Zhong, Gan-Yuan; (Urbandale,
IA) ; Wang, Jimei; (Johnston, IA) ; Mullen,
Jeffrey A.; (Woodward, IA) ; Scelonge, Christopher
J.; (Des Moines, IA) ; Cole, Glenn S.;
(Woodland, CA) ; Soper, John F.; (Urbandale,
IA) |
Correspondence
Address: |
PIONEER HI-BRED INTERNATIONAL INC.
7100 N.W. 62ND AVENUE
P.O. BOX 1000
JOHNSTON
IA
50131
US
|
Family ID: |
22816920 |
Appl. No.: |
09/907411 |
Filed: |
July 17, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60218895 |
Jul 18, 2000 |
|
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|
Current U.S.
Class: |
800/278 ;
435/6.16 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12N 15/8202 20130101; C12Q 1/6855 20130101; C12N 15/8201 20130101;
C12Q 1/6895 20130101; C12N 15/8207 20130101; C12N 15/8209 20130101;
A01H 1/04 20130101; C12N 15/8205 20130101; A01H 5/10 20130101 |
Class at
Publication: |
800/278 ;
435/6 |
International
Class: |
C12Q 001/68; A01H
001/00 |
Claims
We claim:
1. A method of identifying the parental origin of a chromosome
containing transferred DNA comprising: a) crossing a recipient
parent with a donor parent to produce a F1 hybrid plant; b)
transforming at least one cell or tissue of the F1 hybrid plant
with transferred DNA; c) finding at least one transformed F1 hybrid
plant cell containing the transferred DNA; and d) using flanking
DNA of at least one transformed F1 hybrid plant cell to identify
the parental origin of the chromosome containing the transferred
DNA.
2. The method of claim 1, further comprising regenerating a
transgenic plant from the transformed F1 hybrid plant cell
identified as containing the transferred DNA in a chromosome of the
recipient parent.
3. The method of claim 1 wherein the transferred DNA comprises at
least one or combinations of a target site, a targeting cassette, a
functional expression unit, a transposon or a transgene.
4. The method of claim 1 wherein the tissue of the F1 hybrid plant
comprises embryos, cells, cell suspension cultures, callus,
meristems, axillary meristems, leaf discs, or pollen.
5. The method of claim 1, wherein at least one nucleotide of the
flanking DNA of the F1 hybrid, and of at least one nucleotide of
the corresponding region in the recipient parent and/or in the
donor parent, is used one or more times in plasmid rescue, inverse
polymerase chain reaction (PCR), Southern hybridization,
sequencing, Restriction Fragment Length Polymorphism (RFLP), Single
Nucleotide Polymorphism (SNP) or cloning to identify the parental
origin of the chromosome containing the transferred DNA.
6. The method of claim 1 wherein transforming comprises
Agrobacterium-mediated or particle gun transformation.
7. The method of claim 1 wherein the recipient parent is more
recalcitrant to transformation than the donor parent.
8. The method of claim 1 wherein the recipient parent comprises one
or more desired characteristics in disease resistance, insect
resistance, yield, stalk strength, standability, green snap, oil,
phytate, silage, herbicide resistance, starch, carbohydrate, sugar,
sterility, fertility, transgenicly produced protein, amino acid
content, or height than the donor parent.
9. The method of claim 1 wherein the recipient parent and the donor
parent are dicots.
10. The method of claim 9 wherein the dicot is soybean, sunflower,
canola, cotton, alfalfa, potato, sugar beet or safflower.
11. The method of claim 1 wherein the recipient parent and the
donor parent are monocots.
12. The method of claim 11 wherein the monocot is maize, wheat,
sorghum, millet rice, barley, oats or rye.
13. The method of claim 12 wherein the maize is inbred maize.
14. The method of claim 11 wherein the recipient parent is PHTE4,
PHAA0, PHP18, PH05F, PH09B, PHP02, PHJ90, PH24E, PHN46, PHT05,
ASKC27 or PH21T.
15. The method of claim 11 wherein the donor parent is Hi-II, A188,
H99, DAB01, DAB02, or DAB012.
16. A transformed F1 hybrid plant cell of claim 1.
17. The method of claim 1 wherein the donor parent is female.
18. A regenerated transgenic plant of the method of claim 2.
19. The method of claim 2 further comprising backcrossing the first
transgenic plant to the recipient parent, and/or selfing, and/or
introgressing into a closely related species one or more times to
obtain a subsequent transgenic plant.
20. A cell of the first or subsequent transgenic plant of claim
19.
21. A seed of the first or subsequent transgenic plant of claim
19.
22. Pollen of the first or subsequent transgenic plant of claim
19.
23. A plant derived from at least one cell of the first or
subsequent transgenic plant of claim 20.
24. A method of identifying the parental origin of a chromosome
containing transferred DNA comprising: a) crossing a recipient
parent with a donor parent to produce at least one F1 hybrid plant;
b) transforming at least one transformed F1 hybrid plant cell or
tissue of the F1 hybrid plant with a first transferred DNA, wherein
the first transferred DNA comprises a target site; c) finding at
least one cell containing the first transferred DNA; d) using
flanking DNA of the transferred DNA to identify the parental origin
of the chromosome containing the first transferred DNA. e)
regenerating a transgenic plant from a F1 hybrid plant cell
identified as comprising the first transferred DNA in a chromosome
of the recipient parent; f) introgressing, through at least one
backcrossing and selection, the transferred DNA to produce a second
recipient parent containing the transferred DNA; g) crossing the
second recipient parent with a donor parent to produce at least one
F2 hybrid plant; h) transforming at least one cell or tissue of the
F2 hybrid plant with a second transferred DNA; i) introducing a
recombinase that recognizes and implements recombination; and j)
finding at least one transformed F2 hybrid plant cell comprising
the first and the second transferred DNA.
25. The method of claim 24, further comprising using the flanking
DNA of at least one transformed F2 hybrid plant cell to identify
the parental origin of the chromosome containing the transferred
DNA.
26. The method of claim 24, further comprising regenerating a first
transgenic plant from a transformed F2 hybrid plant cell comprising
the first and second transferred DNA in a chromosome of the
recipient parent.
27. The method of claim 26 further comprising backcrossing the
first transgenic plant to the recipient parent, and/or selling,
and/or introgressing into a closely related species one or more
times to obtain a subsequent transgenic plant.
28. The method of claim 24 wherein the first transferred DNA
comprises a targeting cassette.
29. The method of claim 28 wherein the targeting cassette comprises
a nucleotide sequence flanked by at least one non-identical
recombination site and wherein the second transferred DNA is
flanked by non-identical recombination sites corresponding to said
recombination sites contained in the target site of said
chromosome.
30. A method of identifying the parental origin of a chromosome
containing a transgene comprising: a) crossing a recipient parent
with a donor parent to produce at least one F1 hybrid plant; b)
transforming at least one cell or tissue of the F1 hybrid plant
with a transferred DNA; c) finding at least one transformed F1
hybrid plant cell containing the transferred DNA; d) digesting
flanking DNA with restriction enzymes; e) ligating adapters to the
digested flanking DNA; f) amplifying digested flanking DNA of the
transformed F1 hybrid plant cell, and comparable DNA from the donor
parent and/or recipient parent at least once with at least one
vector-specific DNA primer and/or at least one nested primer to
produce a PCR product; and g) sequencing the PCR product or
digesting PCR product with at least one restriction enzyme to
identify parental origin of chromosome.
31. The method of claim 30 wherein the adapters are bubble
adapters.
32. A method of increasing introgression of transferred DNA into a
recipient parent line comprising: a) crossing a recipient parent
with a donor parent to produce at least one F1 hybrid plant; b)
transforming at least one cell or tissue of the F1 hybrid plant
with transferred DNA; c) finding at least one cell containing the
transferred DNA; d) using flanking DNA of at least one transformed
F1 hybrid plant cell to increase introgression of transferred DNA
into recipient parent line.
33. The method of claim 32 wherein the transgenic event frequency
is improved over that of the recipient parent.
34. The method of claim 32 wherein the recipient parent is male and
the donor is female.
35. A method of transforming a plant cell comprising: a) crossing a
male recipient parent with a female donor parent to produce a F1
hybrid plant; b) transforming at least one cell or tissue of the F1
hybrid plant with transferred DNA; and c) finding at least one
transformed F1 hybrid plant cell containing the transferred DNA and
resulting in a transformed F1 hybrid plant cell.
36. The method of claim 35, further comprising using flanking DNA
of at least one transformed F1 hybrid plant cell to identify the
parental origin of the chromosome containing the transferred
DNA.
37. The method of claim 36, wherein at least one nucleotide of the
flanking DNA of the F1 hybrid, and of at least one nucleotide of
the corresponding region in the recipient parent and/or in the
donor parent, is used one or more times in plasmid rescue, inverse
polymerase chain reaction (PCR), Southern hybridization,
sequencing, Restriction Fragement Length Polymorphism (RFLP),
Single Nucleotide Polymorphism (SNP) or cloning to identify the
parental origin of the chromosome containing the transferred
DNA.
38. The method of claim 35, further comprising regenerating a
transgenic plant from the transformed F1 hybrid plant cell
identified as containing the transferred DNA in a chromosome of the
recipient parent.
39. The method of claim 35 wherein the transferred DNA comprises at
least one of or combinations of a target site, a targeting
cassette, a functional expression unit, a transposon or a
transgene.
40. The method of claim 35 wherein the tissue of the F1 hybrid
plant comprises embryos, cells, cell suspension cultures, callus,
meristems, axillary meristems, leaf discs, or pollen.
41. The method of claim 35 wherein transforming comprises
Agrobacterium-mediated or particle gun transformation.
42. The method of claim 35 wherein the recipient parent is more
recalcitrant to transformation than the donor parent.
43. The method of claim 35 wherein the recipient parent comprises
one or more desired characteristics in disease resistance, insect
resistance, yield, stalk strength, standability, green snap, oil,
phytate, silage, herbicide resistance, starch, carbohydrate, sugar,
sterility, fertility, transgenicly produced protein, amino acid
content, or height than the donor parent.
44. The method of claim 35 wherein the recipient parent and the
donor parent are dicots.
45. The method of claim 44 wherein the dicot is soybean, sunflower,
canola, cotton, alfalfa, potato, sugar beet or safflower.
46. The method of claim 35 wherein the recipient parent and the
donor parent are monocots.
47. The method of claim 46 wherein the monocot is maize, wheat,
sorghum, rice, barley, oats or rye.
48. The method of claim 47 wherein the maize is inbred maize.
49. The method of claim 46 wherein the recipient parent is PHTE4,
PHAA0, PHP18, PH05F, PH09B, PHP02, PHJ90, PH24E, PHN46, PHT05,
ASKC27 or PH21T.
50. The method of claim 46 wherein the donor parent is Hi-II, A188,
H99, DAB01, DAB02, or DAB012.
51. The transformed F1 hybrid plant cell of claim 35.
52. The regenerated transgenic plant of the method of claim 38.
53. The method of claim 38 further comprising backcrossing the
first transgenic plant to the recipient parent, and/or selfing,
and/or introgressing into a closely related species one or more
times to obtain a subsequent transgenic plant.
54. A cell of the first or subsequent transgenic plant of claim
53.
55. A seed of the first or subsequent transgenic plant of claim
53.
56. Pollen of the first or subsequent transgenic plant of claim
53.
57. A plant derived from at least one cell of the first or
subsequent transgenic plant of claim 54.
58. A method of increasing survival and/or seed production of a T0
plant comprising: a) crossing a recipient parent with a donor
parent to produce a F1 hybrid plant; b) transforming at least one
cell or tissue of the F1 hybrid plant with transferred DNA; d)
finding at least one transformed F1 hybrid plant cell containing
the transferred DNA; and e) regenerating a transgenic plant from
the transformed F1 hybrid plant cell containing the transferred DNA
to increase survival and seed production of a T0 plant when
compared to survival and seed production of the recipient parent.
Description
CROSS-REFERENCE TO OTHER APPLICATIONS
[0001] This application claims priority to U.S. patent application
60/218,895 filed Jul. 18, 2000 the disclosure of which is
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of genetic
engineering of plants and to methods for introducing traits into
plants.
SUMMARY OF THE INVENTION
[0003] Methods for plant transformation, for improving
transformation efficiency, and for producing desired transgenic
plants are provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] FIG. 1: Restriction digestion analysis of the clone 19-2
containing a flanking sequence from TC1; TC1 was co-transformed
with the plasmids PHP9 and PHP7, containing pat, which have no Mlu
I restriction sites. The presence of a Mlu I restriction site in
the clone 19-2 suggested that the clone contained a DNA sequence
from host genomic.
[0005] FIG. 2: Southern hybridization patterns of Hi-II, PHN46, and
the transgenic Ht12 event TC1 as revealed by probing the DNAs with
a genomic sequence flanking the transgenic DNA of TC1; the Southern
profile suggested that the Ht12 gene was integrated into a Hi-II
chromosome in the event.
[0006] FIG. 3: Comparisons of the sequence profiles of the clone
28, Hi-II, PHN46, and the transgenic event TC0 in the genomic
regions flanking transgenic Ht12 DNA of TC0. The sequence profiles
suggested that the transgene was integrated into a Hi-II
chromosome. SNPs were indicated by dots.
[0007] FIG. 4: Inverse-PCR cloning of host genomic sequences
flanking a transgene. FIG. 4A provides primary PCR amplification
while 4B shows a nested PCR sample of 4A; Column 1 shows the
negative control and Column 2 shows transgenic event 2482.53
1-12A.
[0008] FIG. 5: Southern hybridization patterns of Hi-II, PHN46, and
the F1 transgenic milps event 2482.53-1-12A as revealed by probing
the DNAs with a genomic sequence flanking right border of the
transgenic DNA of 2482.53-1-12A; the Southern hybridization
profiles suggested that the milps gene was integrated into a PHN46
chromosome in the event.
[0009] FIG. 6: Sequence comparisons of the clone JR451, Hi-II,
PHN46, and the transgenic event 2482.53-1-12B in the genomic
regions flanking transgenic milps DNA of 2482.53-1-12B. The
sequence profiles suggested that the milps gene was integrated into
a PHN46 chromosome in the event. SNPs were indicated by dots.
[0010] FIG. 7: Bubble Adaptors
DETAILED DESCRIPTION OF THE INVENTION
[0011] Methods for improving transformation and transformation
efficiency in a plant are provided, especially with regard to
introgression of a transferred DNA into a chosen inbred. The
methods comprise sexually crossing a recipient parent plant chosen
from a genetic line of a plant species of interest with a donor
parent plant selected from a more transformation-competent genetic
line of the plant species, or of another closely related plant
species, to obtain an F1 hybrid progeny. An F1 hybrid can also be
obtained through non-sexual means and is not limited to in-vivo
techniques. A cell obtained from the F1 hybrid progeny can then be
transformed with one or more nucleotide sequences of interest. The
donor parent plant typically is highly transformable but could be
of limited, or perhaps deleterious, breeding value. The recipient
parent plant typically is a recalcitrant inbred used in product
development that exhibits no or poor transformability. When the F1
hybrid between recipient plant and donor plant is made, the
disparate phenotypes of the parents are reconciled to produce cells
and a plant with a hybrid phenotype consisting of higher
transformability than the recipient parent. In addition, the F1
hybrid phenotype offers the potential to introduce a transgene into
a chromosome derived from the recipient plant. The DNA flanking the
transgene can be used to identify the parental origin of the
chromosome containing the transgene, and transgenic events
identified as containing transgenes in the chromosome from the
recipient parent plant are regenerated.
[0012] By "improving transformation efficiency" is intended that
the number of transformed plants recovered per unit time and per
unit resource is increased at least about two-fold, at least about
five-fold, or at least about ten-fold. It is recognized that in
some instances, particularly in inbred plants, particularly those
inbreds exhibiting little or no transformation capability,
transformation is made possible by the methods of the present
invention. The methods of the invention provide a more efficient
means for introducing foreign DNA into plants, particularly inbred
lines of plants, more particularly those plant genetic lines that
are recalcitrant to transformation. The invention allows for the
direct insertion of the foreign DNA into a chromosome derived from
the recipient plant with high frequency, thus reducing the number
of backcrosses to obtain a transgenic plant having desired
characteristics of the genetic line from which the recipient parent
plant is derived. Thus, the invention improves the production
efficiency of transformants as well as reduces backcrossing time to
introgress a transgene into a transgenic plant of interest,
particularly an inbred, having the nucleotide sequence of interest
incorporated in its genome.
[0013] By "transformation" is intended the genetic manipulation of
the plant, cell, cell line, callus, tissue, plant part, seed, and
the like. That is, such cell, cell line, callus, tissue, plant
part, seed, or plant has been altered by the presence of
recombinant DNA wherein said DNA is introduced into the genetic
material within the cell. Preferably, the DNA is introduced into a
chromosome. Recombinant DNA includes foreign DNA, heterologous DNA,
exogenous DNA, chimeric DNA, and endogenous DNA wherein said
endogenous DNA has been derived from the natural chromosomal site
within the plant.
[0014] By "recipient parent" is intended a plant selected from a
genetic line of a plant species of interest. Generally, the
recipient parent will be a genotype that is recalcitrant to
transformation, generally an inbred plant, and more particularly a
recalcitrant inbred plant that possesses high breeding value and is
used in development and production of products. By "recalcitrant"
is intended the genotype of plant exhibits a low level of
transformation efficiency relative to the donor parent, and few or
no transgenic events per unit time and resource can be produced.
Thus a recipient parent that is recalcitrant to transformation, in
the current art has a transformation efficiency that is less than
GS3.
[0015] By "donor parent" is intended a plant selected from a
genetic line of a plant species of interest that is transformation
competent, but may be of limited agronomic value. That is, a useful
level of transformation efficiency is observed in the plant, plant
cell or plant part thereby effecting a greater throughput of
transgenic events. Generally, a useful level of transformation
efficiency comprises an efficiency of at least about 5% (720
transgenic events/year/person), often greater than 30% (4300
transgenic events/year/person), more often greater than 20% (2900
transgenic events/year/person), and most often greater than 10%
(1400 transgenic events/year/person). Thus, by "transformation
competent" is intended a level of transformation efficiency of at
least about 5%. In maize, for example, donor parents include but
are not limited to Hi-II, A188, H99, DAB01, DAB02, DAB012, and
other lines that generate type II callus when cultured, and/or that
exhibit high transformation efficiency. The donor parent may be
from the same plant species as the recipient parent, or may be from
another closely related plant species. By "closely related plant
species" is intended the two species are sexually compatible, that
is, they form viable seed following cross-pollination, and are
capable of producing fertile hybrid progeny.
[0016] By "F1 hybrid" is intended a plant or plant cell resulting
from a cross between a recipient parent of the invention and a
donor parent of the invention. This cross can occur naturally by,
for example, sexual reproduction, or artificially by, for example
in vitro nuclear fusion. The F1 hybrid plants of the invention will
contain approximately one half of the genetic complement of the
donor parent, i.e., that chromosomal material whose ancestral
origin resides with the donor parent, and approximately one half of
the genetic complement of the recipient parent, i.e., that
chromosomal material whose ancestral origin resides with the
recipient parent. For purposes of the present invention, a
chromosome having an ancestral origin residing with a donor parent
is referred to as a "donor chromosome", while a chromosome having
an ancestral origin residing with the recipient parent is referred
to as a "recipient chromosome". Similarly, when reference is made
to a chromosome "derived" from a donor parent or a recipient
parent, it is intended that the chromosome has an ancestral origin
residing with a donor parent or with a recipient parent,
respectively. While the donor parent may be used as either the male
parent or female parent in the cross, it may be preferable to use
the donor parent as the female parent, as a higher transformation
efficiency is observed with this type of cross in some cases.
[0017] By "flanking DNA" is intended DNA genetically and/or
physically linked to the site of integration of a transferred DNA.
Such flanking DNA can range in size from a single base pair to an
entire linear or circular DNA molecule.
[0018] By "amplified" is meant the construction of multiple copies
of a nucleic acid sequence or multiple copies complementary to the
nucleic acid sequence using at least one of the nucleic acid
sequences as a template. Amplification systems include the
polymerase chain reaction (PCR) system, ligase chain reaction (LCR)
system, nucleic acid sequence based amplification (NASBA, Cangene,
Mississauga, Ontario), Q-Beta Replicase systems,
transcription-based amplification system (TAS), and strand
displacement amplification (SDA). See, e.g., Diagnostic Molecular
Microbiology: Principles and Applications, D. H. Persing et al.,
Ed., American Society for Microbiology, Washington, D.C. (1993).
The product of amplification is termed an amplicon.
[0019] By "adapters" is intended small, linear segments of
double-stranded DNA. Such adapters and methods of their synthesis
and use are known to those of ordinary skill in the art. Generally,
such adapters are 5-50 base pairs in length and are prepared by
synthesizing two separate single-stranded DNA molecules that are at
least partially complementary and placing the two molecules
together in conditions that favor the formation of double-stranded
DNA. A "bubble adapter" is a type of adapter that comprises two
single-stranded DNA molecules that when annealed has at least one
internal region that is not complementary.
[0020] As used herein, "nucleic acid" includes reference to a
deoxyribonucleotide or ribonucleotide polymer, or chimeras thereof,
in either single- or double-stranded form, and unless otherwise
limited, encompasses known analogues having the nature of natural
nucleotides in that they hybridize to single-stranded nucleic acids
in a manner similar to naturally occurring nucleotides (e.g.,
peptide nucleic acids).
[0021] By "transferred DNA" is intended at least one nucleotide
sequence that may function to change the phenotype of the plant,
such as a coding sequence for a gene of interest. Particular genes
of interest include those that provide a readily analyzable
functional feature to the host cell and/or organism, such as marker
genes, transgenes as well as other genes that alter the phenotype
of the recipient cells, and the like. Thus, genes affecting plant
growth, height, susceptibility to disease, insects, nutritional
value, oil quality, starch content, glucan content, amino acid
content and the like may be utilized in the invention. The
nucleotide sequence may be a sequence for a gene fragment that can
be targeted for replacement of a fragment of a naturally occurring
gene of interest using gene-targeting methods known in the art. The
nucleotide sequence also may encode an "antisense" sequence to turn
off or modify gene expression. It can include transgenes,
transposable elements and polynucleotides of all lengths and
purpose, coding and non-coding.
[0022] As used herein, the term "plant" includes reference to whole
plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and
plant cells and progeny of same. "Plant cell", as used herein
includes, without limitation, seeds, suspension cultures, embryos,
meristematic regions, callus tissue, leaves, roots, shoots,
gametophytes, sporophytes, pollen, and microspores. The class of
plants which can be used in the methods of the invention include
both monocotyledonous and dicotyledonous plants.
[0023] As used herein, "transgenic plant" includes reference to a
plant which comprises within its genome a heterologous
polynucleotide. Generally, the heterologous polynucleotide is
stably integrated within the genome such that the polynucleotide is
passed on to successive generations. The heterologous
polynucleotide may be integrated into the genome alone or as part
of a recombinant expression cassette. "Transgenic" is used herein
to include any cell, cell line, callus, tissue, plant part or
plant, the genotype of which has been altered by the presence of
heterologous nucleic acid including those transgenics initially so
altered as well as those created by sexual crosses or asexual
propagation from the initial transgenic plant material. The term
"transgenic" as used herein does not encompass the alteration of
the genome (chromosomal or extra-chromosomal) by conventional plant
breeding methods or by naturally occurring events such as random
cross-fertilization, non-recombinant viral infection,
non-recombinant bacterial transformation, non-recombinant
transposition, or spontaneous mutation.
[0024] Recipient genotypes that may function in hybrid combination
with Hi-II, A188 or another donor parent for transformation include
PHTE4, PHAA0, PHP18, PH05F, PH09B, PHP02, PHJ90, PH24E, PHN46,
PHT05, ASKC27 PH21T, PHR8K, PHH3V, PHW0Y, PHG9B, PHK9D, PHT7G,
PHJ9D, PHE8D, PHZ3V, PHZ6C, PHV2E, PHN8Y, PHBE2, PH08A, PH14T,
PH224, PH5WB, Gaspe Flint or one of the following U.S. Pat.
Nos.:
1 6,025,547 Feb. 15, 2000 Inbred maize line PH1CA; 6,020,543 Feb.
1, 2000 Inbred maize line PH1B5; 5,998,711 Dec. 7, 1999 Inbred
Maize Line PH09E 5,990,393 Nov. 23, 1999 Inbred Maize Line PH1CN;
5,986,185 Nov. 16, 1999 Inbred Maize Line PH24D; 5,986,184 Nov. 16,
1999 Inbred Maize Line PH1TB; 5,977,456 Nov. 2, 1999 Inbred Maize
Line PH1M7; 5,977,451 Nov. 2, 1999 Inbred Maize Line PHFW4;
5,948,957 Sep. 7, 1999 Inbred Maize Line PH19V; 5,942,671 Aug. 24,
1999 Inbred Maize Line PH185; 5,942,670 Aug. 24, 1999 Inbred Maize
Line PH14T; 5,939,608 Aug. 17, 1999 Inbred Maize Line PH080;
5,939,607 Aug. 17, 1999 Inbred Maize Line PH2CB; 5,936,148 Aug. 10,
1999 Inbred Maize Line PH1GC; 5,929,313 Jul. 27, 1999 Inbred Maize
Line PHMJ2; 5,917,134 Jun. 29, 1999 Inbred Maize Line PHDN7;
5,917,125 Jun. 29, 1999 Inbred Maize Line PHO3D; 5,889,188 Mar. 30,
1999 Inbred Maize Line PH0B4; 5,866,768 Feb. 2, 1999 Inbred Maize
Line PH02T; 5,866,767 Feb. 2, 1999 Inbred Maize Line PH79A;
5,859,354 Jan. 12, 1999 Inbred Maize Line PH09B; 5,859,316 Jan. 12,
1999 Inbred Maize Line PH0HR; 5,859,313 Jan. 1, 1999 Inbred Maize
Line PHKV0; 5,850,010 Dec. 15, 1998 Inbred Maize Line PH56C;
5,850,009 Dec. 15, 1998 Inbred Maize Line PH0HC; 5,850,007 Dec. 15,
1998 Inbred Maize Line PH1MR; 5,844,117 Dec. 1, 1998 Inbred Maize
Line PH0GP; 5,844,116 Dec. 1, 1998 Inbred Maize Line PH1W2;
5,841,015 Nov. 24, 1998 Inbred Maize Line PH05G; 5,824,847 Oct. 20,
1998 Inbred Maize Line PH22G; 5,824,845 Oct. 20, 1998 Inbred Maize
Line PH10A; 5,811,637 Sep. 22, 1998 Inbred Maize Line PH40B;
5,792,915 Aug. 11, 1998 Inbred Maize Line PH0AV; 5,792,912 Aug. 11,
1998 Inbred Maize Line PH00M; 5,792,911 Aug. 11, 1998 Inbred Maize
Line PH24M; 5,770,790 Jun. 23, 1998 Inbred Maize Line PH41E;
5,767,340 Jun. 16, 1998 Inbred Maize Line PHBR2; 5,763,757 Jun. 9,
1998 Inbred Maize Line PH07D; 5,763,746 Jun. 9, 1998 Inbred Maize
Line PH20A; 5,763,744 Jun. 9, 1998 Inbred Maize Line PH67A;
5,763,743 Jun. 9, 1998 Inbred Maize Line PH63A; 5,750,849 May. 12,
1998 Inbred Maize Line PH05W; 5,750,847 May. 12, 1998 Inbred Maize
Line PH38D; 5,750,835 May. 12, 1998 Inbred Maize Line PH47A;
5,750,834 May. 12, 1998 Inbred Maize Line PH80B; 5,750,832 May. 12,
1998 Inbred Maize Line PH44G; 5,750,831 May. 12, 1998 Inbred Maize
Line PH25A; 5,750,830 May. 12, 1998 Inbred Maize Line PH15A;
5,750,829 May. 12, 1998 Inbred Maize Line PH0AA; 5,731,493 Mar. 24,
1998 Inbred Maize Line PH63B; 5,731,492 Mar. 24, 1998 Inbred Maize
Line PH19A; 5,731,491 Mar. 24, 1998 Inbred Maize Line PHNG2;
5,728,919 Mar. 17, 1998 Inbred Maize Line PHBF0; 5,723,723 Mar. 3,
1998 Inbred Maize Line PH44A; 5,723,722 Mar. 3, 1998 Inbred Maize
Line PHND1; 5,708,189 Jan. 13, 1998 Inbred Corn Line PHP38;
5,675,066 Oct. 17, 1997 Inbred Maize Line PH06N; 5,639,946 Jun. 17,
1997 Inbred Maize Line PHDP0; 5,633,427 May. 27, 1997 Inbred Corn
Line PPHHB4; 5,625,133 Apr. 29, 1997 Inbred Maize Line PH0C7;
5,625,132 Apr. 29, 1997 Inbred Maize Line PH08B; 5,625,129 Apr. 29,
1997 Inbred Corn Line PHDD6; 5,618,987 Apr. 8, 1997 Inbred Maize
Line PH42B; 5,608,140 Mar. 4, 1997 Inbred Maize Line PH38B;
5,608,139 Mar. 4, 1997 Inbred Maize Line PH05F; 5,608,138 Mar. 4,
1997 Inbred Maize Line PHKV1; 5,602,318 Feb. 11, 1997 Inbred Maize
Line PHDG1; 5,602,317 Feb. 11, 1997 Inbred Maize Line PHAA0;
5,569,822 Oct. 29, 1996 Inbred Maize Line PHTE4; 5,569,821 Oct. 29,
1996 Inbred Corn Line PHT11; 5,569,819 Oct. 29, 1996 Inbred Maize
Line PHPP8; 5,569,818 Oct. 29, 1996 Inbred Maize Line PHAP8;
5,569,817 Oct. 29, 1996 Inbred Maize Line PHJJ3; 5,569,816 Oct. 29,
1996 Inbred Maize Line PHAJ0; 5,567,861 Oct. 22, 1996 Inbred Corn
Line PHN46; 5,563,325 Oct. 8, 1996 Inbred Maize Line PHBE2;
5,563,322 Oct. 8, 1996 Inbred Maize Line PHAG6; 5,563,321 Oct. 8,
1996 Inbred Maize Line PHGF5; 5,563,320 Oct. 8, 1996 Inbred Maize
Line PH54B; 5,557,038 Sep. 17, 1996 Inbred Maize Line PHTP9;
5,557,034 Sep. 17, 1996 Inbred Corn Line PHN18; 5,545,814 Aug. 13,
1996 Inbred Maize Line PHFR8; 5,545,813 Aug. 13, 1996 Inbred Maize
Line PHRF5; 5,545,812 Aug. 13, 1996 Inbred Maize Line PHNJ2;
5,545,809 Aug. 13, 1996 Inbred Maize Line PHBG4; 5,543,575 Aug. 6,
1996 Inbred Corn Line PHK46; 5,541,352 Jul. 30, 1996 Inbred Corn
Line PHRD6; 5,534,661 Jul. 9, 1996 Inbred Maize Line PHKW3;
5,530,184 Jun. 25, 1996 Inbred Maize Line PHAP1; 5,527,986 Jun. 18,
1996 Inbred Corn Line PHTD5; 5,506,368 Apr. 9, 1996 Inbred Corn
Line PHN82; 5,506,367 Apr. 9, 1996 Inbred Corn Line PHP38;
5,495,069 Feb. 27, 1996 Inbred Corn Line PHTE4; 5,495,065 Feb. 27,
1996 Inbred Corn Line PHW06; 5,491,286 Feb. 13, 1996 Inbred Corn
Line PHKM5; 5,476,999 Dec. 19, 1995 Inbred Corn Line PHR63;
5,463,173 Oct. 31, 1995 Inbred Corn Line PHR61; 5,453,564 Sep. 26,
1995 Inbred Corn Line PHTE4; 5,444,178 Aug. 22, 1995 Inbred Corn
Line PHHB4; 5,436,390 Jul. 25, 1995 Inbred Corn Line PHR03;
5,434,346 Jul. 18, 1995 Inbred Corn Line PHT11; 5,416,254 May. 16,
1995 Inbred Corn Line PHRE1; 5,387,755 Feb. 7, 1995 Inbred Corn
Line PHFA5; 5,387,754 Feb. 7, 1995 Inbred Corn Line PHGW7;
5,367,109 Nov. 22, 1994 Inbred Corn Line PHHB9; 5,365,014 Nov. 15,
1994 Inbred Corn Line PHMK0; 5,354,942 Oct. 11, 1994 Inbred Corn
Line PHEM9; 5,354,941 Oct. 11, 1994 Inbred Corn Line PHEW7;
5,349,119 Sep. 20, 1994 Inbred Corn Line PHTM9; 5,347,081 Sep. 13,
1994 Inbred Corn Line PHK56; 5,347,080 Sep. 13, 1994 Inbred Corn
Line PHK74; 5,347,079 Sep. 13, 1994 Inbred Corn Line PHGV6;
5,304,720 Apr. 19, 1994 Inbred Corn Line PHHV4; 5,304,719 Apr. 19,
1994 Inbred Corn Line PHT47; 5,285,004 Feb. 8, 1994 Inbred Corn
Line PHBW8; 5,276,265 Jan. 4, 1994 Inbred Corn Line PHR31;
5,245,125 Sep. 14, 1993 Inbred Corn Line PHJ90; 5,220,114 Jun. 15,
1993 Inbred Corn Line PHJ65; 5,159,134 Oct. 27, 1992 Inbred Corn
Line PHP55; 5,159,133 Oct. 27, 1992 Inbred Corn Line PHV37;
5,159,132 Oct. 27, 1992 Inbred Corn Line PHR63; 5,157,208 Oct. 20,
1992 Inbred Corn Line PHN73; 5,157,206 Oct. 20, 1992 Inbred Corn
Line PHN82; 5,097,096 Mar. 17, 1992 Inbred Corn Line PHW20;
5,097,095 Mar. 17, 1992 Inbred Corn Line PHM10; 5,097,094 Mar. 17,
1992 Inbred Corn Line PHP60; 5,097,093 Mar. 17, 1992 Inbred Corn
Line PHJ33; 5,097,092 Mar. 17, 1992 Inbred Corn Line PHR62;
5,095,174 Mar. 10, 1992 Inbred Corn Line PHK35; 5,082,992 Jan. 21,
1992 Inbred Corn Line PHP02; 5,082,991 Jan. 21, 1992 Inbred Corn
Line PHN37; 4,812,600 Mar. 14, 1989 Inbred Corn Line PHK29;
4,812,599 Mar. 13, 1989 Inbred Corn Line PHV78; 4,806,669 Feb. 21,
1989 Inbred Corn Line PHK05; 4,806,652 Feb. 21, 1989 Inbred Corn
Line PHR25;
[0025] and these U.S. patent applications are incorporated by
reference.
[0026] Once F1 hybrid seed has been obtained, tissue from the seed
may be utilized for transformation or the F1 hybrid seed may be
germinated and tissue from the F1 plant utilized for
transformation. In another embodiment, the F1 may be selfed or
crossed with either the recipient or donor parent and F2 or
subsequent generations of embryos or tissue utilized in this
invention. By "tissue" is intended, for example, embryos, cells,
cell suspension cultures, callus, meristems, axillary meristems,
leaf discs, and pollen.
[0027] The methods of the invention can be practiced with any
plant. Such plants include but are not limited to maize (Zea mays),
canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago
sativa), rice (Oryza sativa), rye (Secale cereale), sorghum
(Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuus),
wheat (Triticum aestivum), soybean (Glycine max), tobacco
(Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis
hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea
batatus), cassava (Manihot esculenta), sugar beets (Beta vulgaris),
millet, oats, barley, wheat, vegetables, ornamentals, Lupinus
albus, Lupinus angustifolius and conifers. Plants of the present
invention may be crop plants (e.g., maize, alfalfa, sunflower,
canola, soybean, cotton, peanut, sorghum, wheat, tobacco etc.) and
rice. They may also be cereals or forage. Of interest are soybeans,
rice, wheat and corn. Corn, in particular, has a number of
recalcitrant inbred lines, which have been bred for use in
producing hybrids of improved agronomic importance but are
difficult to transform. Although it would be beneficial to be able
to insert a DNA sequence of interest directly into these inbred
lines, transformation in such lines is difficult or a rare event.
The present invention provides a means for increasing
transformation efficiency as well as reducing the length of time to
obtain the gene in a recalcitrant inbred line.
Genes of Interest
[0028] Genes of interest are reflective of the commercial markets
and interests of those involved in the development of the crop.
Crops and markets of interest change, and as developing nations
open up world markets, new crops and technologies will emerge also.
In addition, as our understanding of agronomic traits and
characteristics such as yield and heterosis increases, the choice
of genes for transformation will change accordingly. General
categories of genes of interest 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 agronomic traits,
insect resistance, disease resistance, herbicide resistance,
sterility, grain characteristics, and commercial products. Genes of
interest include, generally, those involved in oil, starch,
carbohydrate, or nutrient metabolism as well as those affecting for
example kernel size, sucrose loading, and the like. The quality of
grain is reflected in traits such as levels and types of oils,
saturated and unsaturated, quality and quantity of essential amino
acids, and levels of cellulose.
[0029] Grain traits such as oil, starch, and protein content can be
genetically altered in addition to using traditional breeding
methods. Modifications include increasing content of oleic acid,
saturated and unsaturated oils, increasing levels of lysine and
sulfur, providing essential amino acids, and also modification of
starch. Hordothionin protein modifications are described in U.S.
Pat. No. 5,990,389 issued Nov. 23, 1999, U.S. Pat. No. 5,885,801
issued Mar. 23, 1999, U.S. Pat. No. 5,885,802 issued Mar. 23, 1999
and U.S. Pat. No. 5,703,049 issued Dec. 30, 1997; herein
incorporated by reference. Another example is lysine and/or sulfur
rich seed protein encoded by the soybean 2S albumin described in
U.S. Pat. No. 5,850,016 issued Dec. 15, 1998, and the chymotrypsin
inhibitor from barley, Williamson et al. (1987) Eur. J. Biochem.
165:99-106, the disclosures of which are herein incorporated by
reference.
[0030] Derivatives of the coding sequences 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 which is incorporated
herein by reference. Other proteins include methionine-rich plant
proteins such as from corn (Pedersen et al. (1986) J. Biol. Chem.
261:6279; Kirihara et al. (1988) Gene 71:359; both of which are
herein incorporated by reference); and rice (Musumura et al. (1989)
Plant Mol. Biol. 12:123, herein incorporated by reference). Other
genes encode latex, Floury 2, growth factors, seed storage factors,
and transcription factors.
[0031] 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 toxic protein 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.
[0032] Genes encoding disease resistance traits include
detoxification genes, such as against fumonosin (U.S. Pat. No.
5,792,931, issued Aug. 11, 1998); avirulence (avr) and disease
resistance 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.
[0033] Herbicide resistance traits may include genes coding for
resistance to herbicides that 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), genes coding for resistance to herbicides that 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, the
nptII gene encodes resistance to the antibiotics kanamycin and
geneticin, and the ALS gene encodes resistance to the herbicide
chlorsulfuron.
[0034] Sterility genes can also be encoded in an expression
cassette and provide an alternative to physical detasseling.
Examples of genes used in such ways include male tissue-preferred
genes and genes with male sterility phenotypes such as QM,
described in U.S. Pat. No. 5,583,210. Other genes include kinases
and those encoding compounds toxic to either male or female
gametophytic development.
[0035] Commercial traits can also be encoded on a gene or genes
that could increase for example, starch for ethanol production, or
provide expression of proteins. Another 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 (polyhydroxybutyrate synthase)
and acetoacetyl-CoA reductase (see Schubert et al. (1988) J.
Bacteriol. 170:5837-5847) facilitate expression of
polyhyroxyalkanoates (PHAs).
[0036] Genes of medicinal and pharmaceutical uses, such as that
encoding avidin and vaccines or proteins produced utilizing plants
as factories are also contemplated as part of this invention.
Gene Targeting
[0037] It is recognized that the nucleotide sequence of interest
also encompasses nucleotide sequences that may be useful in
manipulating the genetic content of a plant, such as sites for
insertion of DNA into the genome. Although such nucleotide
sequences may not change the phenotype of the plant, the genetic
material of the plant will be altered. Insertion or recombination
sites for use in the invention are known in the art and include FRT
sites (see, for example, Schlake et al. (1994) Biochemistry
33:12746-12751; Huang et al. (1991) Nucleic Acids Res. 19:443-448;
Sadowski (1995) Prog. Nuc. Acid Res. Mol. Bio. 51:53-91; Cox (1989)
Mobile DNA, ed. Berg and Howe (American Society of Microbiology,
Washington D.C.), pp. 116-670; Dixon et al. (1995) 18:449-458;
Umlauf et al. (1988) EMBO J. 7:1845-1852; Buchholz et al. (1996)
Nucleic Acids Res. 24:3118-3119; Kilby et al. (1993) Trends Genet.
9:413-421: Roseanne et al. (1995) Nat. Med. 1:592-594; Albert et
al. (1995) Plant J. 7:649-659: Bailey et al. (1992) Plant Mol.
Biol. 18:353-361; Odell et al. (1990) Mol. Gen. Genet. 223:369-378;
and Dale et al. (1991) Proc. Natl. Acad. Sci. USA 88:10558-105620;
all of which are herein incorporated by reference); and lox (Albert
et al. (1995) Plant J. 7:649-659; Qui et al. (1994) Proc. Natl.
Acad. Sci. USA 91:1706-1710; Sturman et al. (1996) Plant Mol. Biol.
32:901-913; Odell et al. (1990) Mol. Gen. Genet. 223:369-378; Dale
et al. (1990) Gene 91:79-85; and Bayley et al. (1992) Plant Mol.
Biol. 18:353-361). Such recombination sites, in the presence of a
compatible recombinase, allow for the targeted integration of one
or more nucleotide sequences of interest into the plant genome.
[0038] Non-identical recombination sites that have been introduced
into the genome of the recipient plant can be used to establish a
target site within a chromosome derived from a recipient plant of
the invention for subsequent insertion of one or more nucleotide
sequences of interest into the recipient chromosome.
[0039] For example, a nucleotide sequence flanked by two
non-identical recombination sites is introduced into a chromosome
derived from the recipient plant (i.e., a recipient chromosome)
thereby establishing a target site for targeted insertion of one or
more nucleotide sequences of interest. By "non-identical"
recombination sites is intended that the flanking recombination
sites are not identical in sequence. That is, one flanking
recombination site may be an FRT site where the second
recombination site may be a mutated FRT site. The non-identical
recombination sites used in the methods of the invention prevent or
greatly suppress recombination between the two flanking
recombination sites and excision of the nucleotide sequence
contained therein. Accordingly, it is recognized that any suitable
non-identical recombination sites may be utilized in the invention,
including FRT and mutant FRT sites, FRT and lox sites, lox and
mutant lox sites, as well as other recombination sites known in the
art.
[0040] By "suitable" non-identical recombination site is intended
that in the presence of active recombinase, excision of sequences
between two non-identical recombination sites occurs, if at all,
with an efficiency considerably lower than the
recombinationally-mediated exchange that targets arrangement of
nucleotide sequences into the plant genome. Thus, suitable
non-identical sites for use in the invention include those sites
where the efficiency of recombination between the sites is low; for
example, where the efficiency is less than about 30 to about 50%,
or less than about 10 to about 30%, or less than about 5 to about
10%.
[0041] In this manner, suitable non-identical recombination sites
are introduced into a chromosome derived from a recipient plant
from a genetic line of a plant species of interest, establishing a
target site for integration of a nucleotide sequence of interest,
such as a transgene, into the recipient chromosome. Introduction of
the non-identical recombination sites, and hence establishment of
the target site, may occur either by traditional breeding practice,
such as with cross-pollination techniques, or by direct
transformation of the hybrid tissue obtained from an F1 hybrid
plant or seed of the invention.
[0042] Thus, in one embodiment of the invention, an initial
recipient plant chosen from a genetic line of the plant species of
interest is sexually crossed with a plant that has within its
genome such non-identical recombination sites flanking a selectable
marker gene, resulting in a hybrid plant. The plant having the
non-identical recombination sites within its genome may be a donor
plant of the invention, i.e., one that is selected from a
transformation competent genetic line of the same plant species or
of another closely related plant species, a plant whose
transformation competence is unknown and is chosen from the same
plant species or another closely related plant species, or a plant
that is recalcitrant to transformation and is chosen from the same
plant species or another closely related plant species. The hybrid
plant resulting from this sexual cross can then be backcrossed with
a second recipient plant from the genetic line of the plant species
of interest to obtain progeny. These progeny can then be screened,
using methods of this invention, for the presence of the target
site (which comprises the non-identical recombination sites
flanking the selectable marker gene) integrated into a chromosome
derived from the recipient plant. Progeny identified as having the
target site integrated into a recipient chromosome can then be used
in a traditional breeding approach to introgress, through repeated
backcrossing and selection, the selectable marker gene and flanking
recombination sites (i.e., the target site) into the genetic line
from which the initial recipient plant was chosen.
[0043] Thus one can obtain a recipient plant having the
recombination sites, and hence the target site for subsequent
integration of a nucleotide sequence of interest, incorporated
within its genome and having a genetic complement similar to other
plants within the genetic line from which the initial recipient
plant was chosen. The resulting recipient plant may, however,
exhibit the low transformation efficiency that is characteristic of
other members of this genetic line. Improved transformation
efficiency is then achieved using methods of the invention. In this
manner, the resulting recipient plant is crossed with a donor plant
as described herein to obtain an F1 hybrid plant or seed of the
invention. This F1 hybrid plant or seed comprises the non-identical
recombination sites integrated into a chromosome derived from the
recipient plant and is characterized by having improved
transformation efficiency relative to the recipient plant, or, in
some instances, relative to both the recipient plant and the donor
plant. The incorporated non-identical recombination sites provide a
target site for integration of other nucleotide sequences of
interest using a targeting cassette in the presence of a compatible
recombinase as described below.
[0044] Alternatively, having identified progeny having the target
site integrated within a recipient chromosome, one may bypass the
introgression step and instead sexually cross the selected progeny
with a donor plant of the invention to obtain an F1 hybrid plant or
seed of the invention characterized by improved transformation
efficiency. As before, the F1 hybrid plant or seed possesses the
target site integrated into a recipient chromosome. Targeted
integration of one or more nucleotide sequences of interest into
the target site of the recipient chromosome is then accomplished
using a targeting cassette in the presence of a compatible
recombinase as described below. Following targeted integration, the
DNA sequences of interest can be introgressed into the genetic line
from which the initial recipient plant was chosen, into another
genetic line of the same plant species or of another closely
related plant species.
[0045] In another embodiment of the invention, the target site is
established in a recipient chromosome by transformation. In this
manner, the hybrid tissue obtained from an F I hybrid plant or seed
of the invention is transformed with a DNA construct comprising the
non-identical recombination sites. Thus, for example, using
transformation techniques well known in the art, the hybrid tissue
is transformed with an expression cassette comprising the
non-identical recombination sites, which in turn flank a selectable
marker gene. Any selectable marker gene may be used so long as
transformed cells can be selected for subsequent culture and plant
regeneration. In this way, transformed cells of the hybrid tissue
having recombination sites incorporated into the genome are
obtained.
[0046] It is recognized that any means of transformation may be
utilized for inserting the recombination sites. However,
Agrobacterium-mediated transformation generally tends to insert a
lower copy number of transferred DNA than does particle bombardment
or other transformation means.
Codon Preference
[0047] Where appropriate, the nucleotide sequences of interest that
are to be stably integrated into the genome of a recipient plant
may be optimized for increased expression in the recipient plant.
Where mammalian, yeast, or bacterial genes are used in the
invention, they can be synthesized using plant-preferred codons for
improved expression. It is recognized that for expression in
monocots, dicot genes can also be synthesized using
monocot-preferred codons. Methods are available in the art for
synthesizing plant-preferred genes. See, for example, U.S. Pat.
Nos. 5,380,831, 5,436,391, and Murray et al. (1989) Nucleic Acids
Res. 17:477-498, herein incorporated by reference.
[0048] The plant-preferred codons may be determined from the codons
utilized more frequently in the proteins expressed in the recipient
plant of interest. It is recognized that monocot-or dicot-preferred
sequences may be constructed as well as plant-preferred sequences
for particular plant species. See, for example, EPA 0359472; EPA
0385962; WO 91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci.
USA 88:3324-3328; and Murray et al. (1989) Nucleic Acids Res.
17:477-498; U.S. Pat. Nos. 5,380,831 and 5,436,391; and the like,
herein incorporated by reference. It is further recognized that all
or any part of the gene sequence may be optimized or synthetic.
That is, fully optimized or partially optimized sequences may also
be used. Additional sequence modifications are known to enhance
gene expression in a cellular host and can be used in the
invention. These include elimination of sequences encoding spurious
polyadenylation signals, exon-intron splice site signals,
transposon-like repeats, and other such well-characterized
sequences, which may be deleterious to gene expression. The G-C
content of the sequence may be adjusted to levels average for a
given cellular host, as calculated by reference to known genes
expressed in the host cell. When possible, the sequence may be
modified to avoid predicted hairpin secondary mRNA structures.
[0049] Thus, for example, where a DNA construct comprising a
compatible recombinase gene is to be used for targeted integration
of a nucleotide sequence of interest into a target site within a
chromosome of interest, the nucleotide sequence encoding the
compatible recombinase may be constructed with plant-preferred
codons. More particularly, where the gene encodes an FLP
recombinase, for example, the FLP gene sequence may be constructed
using plant-preferred codons to obtain an FLP recombinase that is
optimized for expression in the recipient plant. See "Novel Nucleic
Acid Sequence Encoding FLP Recombinase", WO99/27077, which is
incorporated by reference.
Transferred DNA
[0050] It is recognized that the nucleotide sequences of interest
may be utilized in a functional expression unit or cassette. By
"functional expression unit" or "cassette" is intended the
nucleotide sequence of interest is operably linked with a
functional promoter, and in most instances a termination region.
There are various ways to achieve the functional expression unit
within the practice of the invention. In one embodiment of the
invention, the nucleotide sequence of interest is transferred or
inserted into the genome as a functional expression unit.
Alternatively, the nucleotide sequence may be inserted into a site
within the genome that is 3' to a promoter region. In this latter
instance, the insertion of the coding sequence 3' to the promoter
region is such that a functional expression unit is achieved upon
integration.
[0051] For convenience, the nucleotide sequences of interest are
provided in expression cassettes for expression in the recipient
plant. The cassette will include 5' and 3' regulatory sequences
operably linked to a nucleotide sequence of interest. By "operably
linked" is intended a functional linkage between a promoter and a
second sequence, wherein the promoter sequence initiates and
mediates transcription of the DNA sequence corresponding to the
second sequence. Generally, operably linked means that the nucleic
acid sequences being linked are contiguous and, where necessary to
join two protein coding regions, contiguous and in the same reading
frame. The cassette may additionally contain at least one
additional gene or nucleotide sequence of interest to be
co-transformed into the plant. Thus, each nucleic acid sequence
will be operably linked to 5' and 3' regulatory sequences.
Alternatively, the additional gene(s) or nucleotide sequence(s) can
be provided on multiple expression cassettes.
[0052] Such an expression cassette is provided with a plurality of
restriction sites for insertion of the nucleotide sequence of
interest that is to be under the transcriptional regulation of the
regulatory regions. The expression cassette may additionally
contain selectable marker genes.
[0053] The expression cassette will include in the 5'-3' direction
of transcription, a transcriptional and translational initiation
region, a nucleotide sequence of interest, and a transcriptional
and translational termination region functional in plants. The
transcriptional initiation region, the promoter, may be native or
analogous or foreign or heterologous to the plant host.
Additionally, the promoter may be the natural sequence or
alternatively a synthetic sequence. By "foreign" is intended that
the transcriptional initiation region is not found in the native
plant into which the transcriptional initiation region is
introduced.
[0054] While it may be preferable to express the nucleotide
sequences of interest using heterologous promoters, the native
promoter sequences may be used. Such constructs would change
expression levels of any protein encoded by a nucleotide sequence
of interest in the plant or plant cell. Thus, the phenotype of the
plant or plant cell is altered.
[0055] The termination region may be native with the
transcriptional initiation region, may be native with the operably
linked DNA sequence of interest, or may be derived from another
source. Convenient termination regions are available from the
potato proteinase inhibitor (PinII) gene or the Ti-plasmid of A.
tumefaciens, such as the octopine synthase and nopaline synthase
termination regions. See also Guerineau et al. (1991) Mol. Gen.
Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et
al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell
2:1261-1272; Munroe et al (1990) Gene 91:151-158; Ballas et al
(1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987)
Nucleic Acid Res. 15:9627-9639.
[0056] The expression cassettes may additionally contain 5' leader
sequences in the expression cassette construct. Such leader
sequences can act to enhance translation. Translation leaders are
known in the art and include: picornavirus leaders, for example,
EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein
et al. (1989) PNAS USA 86:6126-6130); potyvirus leaders, for
example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986);
MDMV leader (Maize Dwarf Mosaic Virus); Virology 154:9-20), and
human immunoglobulin heavy-chain binding protein (BiP), (Macejak et
al. (1991) Nature 353:90-94); untranslated leader from the coat
protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al.
(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV)
(Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss,
New York), pp. 237-256); and maize chlorotic mottle virus leader
(MCMV) (Lommel et al. (1991) Virology 81:382-385). See also,
Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. Other methods
known to enhance translation can also be utilized, for example,
introns, and the like.
[0057] In preparing the expression cassette, the various DNA
fragments may be manipulated, so as to provide for the DNA
sequences in the proper orientation and, as appropriate, in the
proper reading frame. Toward this end, adapters or linkers may be
employed to join the DNA fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of
superfluous DNA, removal of restriction sites, or the like. For
this purpose, in vitro mutagenesis, primer repair, restriction,
annealing, resubstitutions, e.g., transitions and transversions,
may be involved.
[0058] Generally, the expression cassette will comprise a
selectable marker gene for the selection of transformed cells or
tissues. See generally, Yarranton (1992) Curr. Opin. Biotech.
3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA
89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992)
Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) Operon, pp.
177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell
49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al.
(1989) Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerst et al.
(1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al.
(1990) Science 248:480-483; M. Gossen (1993) Ph.D dissertation,
University of Heidelberg; Reines et al. (1993) Proc. Natl. Acad.
Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell Bio.
10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA
89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA
88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res.
19:4647-4653; Hillenand-Wissman (1989) Topics in Mol. and Struc.
Biol. 10:143-162; Degenkolb et al. (1991) Antimicrob. Agents
Chemother. 35:1591-1595; Kleinschnidt et al. (1988) Biochemistry
27:1094-1104; Gatz et al. (1992) Plant J. 2:397-404; A. L. Bonin
(1993) Ph.D. dissertation, University of Heidelberg; Gossen et al.
(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992)
Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985)
Handbook of Exp. Pharmacology 78; Gill et al. (1988) Nature
334:721-724. Such disclosures are herein incorporated by
reference.
Transformation
[0059] Once the tissue is obtained from the F1 hybrid plant or
seed, the nucleotide sequences of interest can be introduced in the
plant by any method known in the art. In this manner, genetically
modified plants, plant cells, plant tissue, seed, and the like can
be obtained. Transformation protocols as well as protocols for
introducing nucleotide sequences into plants may vary depending on
the type of plant or plant cell, i.e., monocot or dicot, targeted
for transformation. Suitable methods of introducing nucleotide
sequences into plant cells and subsequent insertion into the plant
genome include microinjection (Crossway et al. (1986) Biotechniques
4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad.
Sci. USA 83:5602-5606, Agrobacterium-mediated transformation
(Townsend et al., U.S. Pat. No. 5,563,055), direct gene transfer
(Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic
particle acceleration (see, for example, Sanford et al., U.S. Pat.
No. 4,945,050; Tomes et al. (1995) "Direct DNA Transfer into Intact
Plant Cells via Microprojectile Bombardment," in Plant Cell,
Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and
Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988)
Biotechnology 6:923-926). Also see Weissinger et al. (1988) Ann.
Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science
and Technology 5:27-37 (onion); Christou et al. (1988) Plant
Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology
6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev.
Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.
Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology
8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA
85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563
(maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat.
Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) "Direct DNA
Transfer into Intact Plant Cells via Microprojectile Bombardment,"
in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed.
Gamborg (Springer-Verlag, Berlin) (maize); Klein et al. (1988)
Plant Physiol. 91:440-444 (maize); Fromm et al. (1990)
Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al.
(1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No.
5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci.
USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The
Experimental Manipulation of Ovule Tissues, ed. Chapman et al.
(Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990)
Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor.
Appl. Genet. 84:560-566 (whisker-mediated transformation);
D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation);
Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and
Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al.
(1996) Nature Biotechnology 14:745-750 and U.S. Pat. No. 5,981,840
(maize via Agrobacterium tumefaciens); all of which are herein
incorporated by reference.
[0060] Another useful basic transformation protocol involves a
combination of wounding by particle bombardment, followed by use of
Agrobacterium for DNA delivery, as described by Bidney, et al.,
Plant Mol. Biol., 18: 301-31 (1992). Useful plasmids for plant
transformation include pBin 19. See Bevan, Nucleic Acids Research,
12: 8711-8721 (1984), and hereby incorporated by reference. This
method is preferred for transformation of sunflower plants.
[0061] In general, the intact meristem transformation method
involves imbibing seed for 24 hours in the dark, removing the
cotyledons and root radical, followed by culturing of the meristem
explants. Twenty-four hours later, the primary leaves are removed
to expose the apical meristem. The explants are placed apical dome
side up and bombarded, e.g., twice with particles, followed by
co-cultivation with Agrobacterium. To start the co-cultivation for
intact meristems, Agrobacterium is placed on the meristem. After
about a 3-day co-cultivation period the meristems are transferred
to culture medium with cefotaxime plus kanamycin for the NPTII
selection.
[0062] The split meristem method involves imbibing seed, breaking
of the cotyledons to produce a clean fracture at the plane of the
embryonic axis, excising the root tip and then bisecting the
explants longitudinally between the primordial leaves. The two
halves are placed cut surface up on the medium then bombarded twice
with particles, followed by co-cultivation with Agrobacterium. For
split meristems, after bombardment, the meristems are placed in an
Agrobacterium suspension for 30 minutes. They are then removed from
the suspension onto solid culture medium for three day
co-cultivation. After this period, the meristems are transferred to
fresh medium with cefotaxime plus kanamycin for selection.
[0063] Many transformation protocols may be used in the practice of
the invention to transform a tissue obtained from the hybrid plant
with one or more nucleotide sequences of interest, although
protocols producing single copy events work better with this
invention. Preferably the transformation protocol is the same
protocol, or a protocol that has minor modifications, as the
protocol used in transformation of tissues obtained from the donor
plant. This protocol yields a useful level of transformation
efficiency in tissues obtained from either the F1 hybrid plant or
seed, or from the donor plant. For example, if the donor plant is
Hi-II, the tissue from the hybrid plant will be transformed using
the standard protocol for Hi-II transformation, or a minor
modification thereof. This strategy not only yields a useful level
of transformation efficiency, it also allows for the use of the
same protocol generally regardless of the genetic line used as the
source of the recipient plant, making transformation of multiple
genetic lines a more efficient process.
[0064] Once the DNA sequence of interest has been introduced into
tissue from the hybrid plant, transformed cells are selected and
transgenic plants regenerated using methods well known in the art.
See, for example, McCormick et al. (1986) Plant Cell Reports,
5:81-84; WO97/41228 and U.S. Pat. No. 5,981,840, which are
incorporated by reference.
[0065] For those hybrid tissues engineered to have a target site,
i.e., non-identical recombination sites, within a recipient
chromosome, use of a targeting cassette in the presence of a
compatible recombinase as described above results in insertion of
the nucleotide sequence of interest within a recipient chromosome.
In this case, plants 30 regenerated from transformed cells will
have the nucleotide sequence of interest inserted within a
recipient chromosome. Thus, these transformed cells that also test
positive for transformation with the nucleotide sequence of
interest may be directly transferred to plant regeneration media,
since those have the transferred DNA in the recipient
chromosome.
[0066] For those hybrid tissues not having target sites
incorporated within a recipient chromosome, plants regenerated from
transformed cells will have approximately a 50:50 chance of having
the nucleotide sequence of interest inserted within a recipient
chromosome. In this case, it is preferable, though not required, to
have an early round of selection that discriminates between those
transformation events having the nucleotide sequence of interest
inserted within a recipient chromosome and those events having the
sequence inserted within a donor chromosome. In this manner, only
those transformation events identified as having the nucleotide
sequence inserted within a recipient chromosome using chromosome
localization methods described above are carried forward for
regeneration into plants.
[0067] These regenerated transgenic plants may then be grown to
maturity and sexually crossed with the same transformed strain
("selfed"), or "backcrossed" with another recipient plant chosen
from the genetic line from which the initial recipient plant was
chosen to obtain transgenic plants having desired characteristics
of the recipient plant. Alternatively, the regenerated transgenic
plants may use to "introgress" the nucleotide sequence of interest
into another genetic line of the same plant species or into a
genetic line of another closely related plant species. In this
manner, it is possible to produce a transgenic plant and obtain
transgenic seed for a genetic line that is characterized by low
transformation efficiency.
Cloning Flanking DNA
[0068] A variety of techniques able to distinguish two DNAs are
contemplated in this invention. Genomic DNA is isolated and used to
identify the flanking DNA through cloning as in plasmid rescue,
library screening, PCR-based techniques or any method that obtains
or identified at least some of the polynucleotides of the flanking
region. Utilization of the flanking DNA, compared with other
methods, provides a greater efficiency and speed with which the
parental chromosome can be identified.
[0069] Methods for isolating genomic DNA from organism are known to
those of ordinary skill in the art. While the methods of the
invention do not depend on any particular method for isolating
genomic DNA, it is recognized that the choice of isolation method
will depend on a variety of factors including, the species of
organism, the age of the organism, the specific cells or tissues
selected and the intended use of the genomic DNA. Methods for the
isolation of genomic DNA are generally known in the art and are
disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory
Manual (2.sup.nd ed., Cold Spring Harbor Laboratory Press,
Plainview, N.Y.) and in The Maize Handbook (1994) pp517-653;
Springer-Verlag New York, which are incorporated by reference.
[0070] 1. Plasmid Rescue
[0071] The principles of plasmid rescue for isolating genomic
sequences flanking transferred DNA can be found in Behringer and
Medford (1992) Plant Mol. Biol. Rept. 10:190-198 and Feldmann
(1992) "T-DNA insertion mutagenesis in Arabidopsis seed
infection/transformation," in Methods in Arabidopsis Research, ed.
Koncz et al. (London: World Scientific), pp. 274-289. The presence
of the antibiotic marker gene, amp, from the transformed plasmid
backbones in the events made it possible to isolate flanking
sequences via this approach. This approach was also used because
plasmid-rescue procedures are generally not sensitive, compared
with PCR-based cloning methods, to the presence of structural
rearrangements of transgenic DNA.
[0072] 2. Inverse PCR
[0073] The principles of inverse PCR for isolating genomic
sequences flanking a known or unknown transferred DNA sequence
(e.g. transgenes and transposons) can be found in Gasch et al.
(1992) "Gene isolation with the polymerase chain reaction," Methods
in Arabidopsis Research, ed. Koncz et al. (London: World
Scientific), pp. 342-356, and Britt and Earp (1994) "The polymerase
chain reaction: applications to maize transposable elements," in
The Maize Handbook, ed. Freeling and Walbot (Springer-Verlag, New
York), pp. 586-592. In the present study, sequences flanking
transgenic DNA from transgenic milps events were obtained by using
an inverse-PCR technique.
[0074] Methods for determining chromosomal location of DNA
sequences of interest are known in the art. See, for example,
Leitch et al. (1996) in Plant Molecular Biology, ed. Clark
(Springer-Verlag, Berlin), pp. 461-519, for a discussion regarding
localization of DNA sequences, and the use of sequential
hybridizations with specific gene probes and genomic in situ
hybridization for allocating transgene insertion events to a
parental genome. One such approach entails the use of Genome
Walker.TM. kit sold by Clontech Laboratories, Inc., Palo Alto,
Calif. cloning or PCR of the genomic DNAs flanking the DNA
construct of interest, followed by sequencing or DNA hybridization
and methods known in the art to determine whether the flanking DNA
is of donor or recipient parent genomic origin. See particularly
the copending entitled "METHODS FOR IDENTIFYING DESIRED TRANSGENIC
ORGANISMS" U.S. application Ser. No. 09/696,621, which is
incorporated by reference in its entirety.
[0075] The flanking DNA can be used to identify the parental origin
of the chromosome containing the transferred DNA. This can be done
through comparison of the F1 hybrid plant, the recipient plant and
the donor plant using for example sequence analysis, Southern
analysis, gel electrophoresis, RFLP, SNP or other means of
identification or combinations thereof.
[0076] One embodiment includes isolating genomic DNA and digesting
with at least one restriction enzyme. Compatible adapters are
ligated to the digested genomic DNA, followed by PCR amplification
employing two primers, one designed to hybridize to a nucleotide
sequence within the adapter and the other designed to hybridize to
a nucleotide sequence within the transgene. The expected product of
such a PCR amplification is a DNA molecule containing a portion of
the transgene and flanking DNA. The nucleotide sequence of the PCR
product can be determined and used to design PCR primers and
hybridization probes specific to the portion of flanking DNA in the
PCR product. Alternatively, the PCR product can be labeled, during
or after amplification, for use as a hybridization probe. Genomic
DNA is then isolated from an ancestor of the transgenic plant and
the PCR product is used directly or indirectly to determine if the
transgene integrated into DNA originating in the donor plant or
recipient plant by any means known to those of ordinary skill in
the art. Typically, such means include a comparison of results
obtained with genomic DNA from one or more ancestors and genomic
DNA from the FI hybrid plant.
[0077] In another embodiment, restriction digested genomic DNA of a
transformed plant is ligated with bubble adapters (FIG. 7). The
ligated genomic DNA is PCR amplified employing two ligand-labeled
primers, one complementary to the bubble region of the bubble
adapter and the other complementary to the transferred DNA. While
any ligand known to those of ordinary skill in the art can be
employed to label the primers, the preferred ligand of the present
invention is biotin. Methods of synthesizing and using
ligand-labeled primers are known to those of ordinary skill in the
art. The PCR product is digested with a restriction enzyme that
cleaves the PCR product into at least three fragments. Then the
ligand-labeled DNA fragments are isolated by methods known to those
of ordinary skill in the art such as, for example, affinity
chromatography. Preferably, single-stranded, ligand-labeled
fragments are isolated. The single-stranded, ligand-labeled
fragments are then used as primers in separate PCR amplification of
genomic DNA from the F1 hybrid plant and one or both parents. The
resulting products of the separate PCR amplifications are compared
to determine the parental origin of the flanking DNA.
[0078] If the transferred DNA contains a selectable marker gene for
use in microorganisms such as bacteria and yeast, plasmid rescue
can be employed instead of inverse PCR. The product of inverse PCR
or the "rescued" plasmid can be used as a hybridization probe for
Southern blotting of restriction enzyme-digested genomic DNA of the
F1 hybrid plant and one or both parents. Or the product can be
analyzed through sequencing, enzyme restriction analysis or other
molecular comparison means.
[0079] The methods of the invention involve identifying at least
one base pair difference between the donor parent and the recipient
parent in the flanking DNA. It may be necessary to identify more
than one distinct DNA fragment comprising flanking DNA.
[0080] While certain embodiment of the invention comprise isolating
genomic DNA from a parent, it is recognized that parental genomic
DNA sequences may have already been determined. It is also
recognized that other ancestors may substitute for the donor and
recipient parents.
[0081] For example, in one embodiment, the initial recipient plant
from the genetic line of a plant species of interest is crossed
with a donor plant that to obtain an F1 hybrid plant or seed of the
invention. Hybrid tissue obtained from this F1 hybrid plant or
seed, which has improved transformation efficiency, can then be
transformed with a DNA construct comprising one or more nucleotide
sequences of interest. Transformed cells of the hybrid tissue
identified as having the transferred DNA within a recipient
chromosome are determined at this point by methods of this
invention and carried forward. In this manner, transformed hybrid
tissue having the nucleotide sequence or sequences of interest
integrated within a donor chromosome is obtained. Following
regeneration of the transformed hybrid tissue, the resulting
transformed plant can then be backcrossed with a second recipient
plant from the genetic line from which the initial recipient plant
was chosen to obtain progeny. Those progeny comprising the
nucleotide sequence of interest within a recipient chromosome can
then be selected for subsequent use in introgressing the nucleotide
sequence of interest into the genetic line from which the initial
recipient plant was chosen, into another genetic line of the plant
species of interest, or into a genetic line of another closely
related plant species of interest. Progeny may also be selfed.
[0082] For example, in one embodiment, the initial recipient plant
from the genetic line of a plant species of interest is crossed
with a donor plant that comprises the target site integrated within
its genome to obtain an F1 hybrid plant or seed of the invention
comprising the target site integrated within a donor chromosome.
Hybrid tissue obtained from this F1 hybrid plant or seed, which has
improved transformation efficiency, can then be transformed with a
DNA construct comprising one or more nucleotide sequences of
interest using a targeting cassette in the presence of a compatible
recombinase as described below. In this manner, transformed hybrid
tissue having the nucleotide sequence or sequences of interest
integrated within a donor chromosome is obtained. Following
regeneration of the transformed hybrid tissue, the resulting
transformed plant can then be backcrossed with a second recipient
plant from the genetic line from which the initial recipient plant
was chosen to obtain progeny. Those progeny comprising the
nucleotide sequence of interest within a recipient chromosome can
then be selected for subsequent use in introgressing the nucleotide
sequence of interest into the genetic line from which the initial
recipient plant was chosen, into another genetic line of the plant
species of interest, or into a genetic line of another closely
related plant species of interest.
[0083] Having established a target site integrated within a
chromosome of interest, which may be a donor chromosome in some
embodiments or a recipient chromosome in other embodiments
described herein, nucleotide sequences of interest can be targeted
for insertion into the target site using a targeting cassette as
described below. In the presence of a compatible recombinase, the
non-identical recombination sites in the chromosome of interest
provide a means of moving desired genes or nucleotide sequences
from the targeting cassette into the chromosome at the location of
the target site.
[0084] It is recognized that the chromosome of interest may
comprise multiple target sites; i.e., sets of non-identical
recombination sites. In this manner, multiple manipulations of the
target site in the plant are available. By "target site" in the
plant is intended the DNA sequence that has been inserted into a
chromosome of interest and which comprises the non-identical
recombination sites. Preferably the target site has been
established within a recipient chromosome so that integration of
the nucleotide sequence of interest can be directed to a recipient
chromosome.
[0085] In one embodiment of the invention, a hybrid tissue obtained
from an F1 hybrid plant or seed of the invention contains at least
one target site integrated into a chromosome derived from the
recipient plant. The target site is characterized by being flanked
by non-identical recombination sites as previously defined.
[0086] By "targeting cassette" is intended a DNA construct
comprising one or more nucleotide sequences of interest flanked by
identical or non-identical recombination sites corresponding to
those non-identical recombination sites contained in the target
site of the chromosome of interest. By "compatible recombinase" is
intended a recombinase that recognizes the non-identical
recombination sites and catalyzes site-specific recombination.
Thus, introduction of the targeting cassette in the presence of a
compatible recombinase results in exchange of the nucleotide
sequence flanked by the non-identical recombination sites of the
target site with the nucleotide sequences of interest from the
targeting cassette such that the nucleotide sequences of interest
now reside on the chromosome of interest. In one embodiment of the
invention, introduction of the targeting cassette in the presence
of a compatible recombinase results in targeted integration of a
nucleotide sequence of interest into a recipient chromosome within
the genome of an F1 hybrid plant or seed of the invention.
[0087] The targeting cassette may be introduced into a plant
comprising the target site integrated within its genome either by
traditional breeding methods or by transformation. Thus, for
example, in one embodiment of the invention, an F1 hybrid plant or
seed comprising a target site integrated within its genome,
preferably within a recipient chromosome, is sexually crossed with
a plant comprising the targeting cassette integrated within its
genome. The latter plant may be from the same plant species as the
F1 hybrid or from another closely related plant species. The
resulting progeny have a target site and a targeting cassette
integrated within their genome. In the presence of an active
compatible recombinase, the nucleotide sequence of interest flanked
by the non-identical sites of the targeting cassette is integrated
into the target site residing on the recipient chromosome. In this
manner, nucleotide sequences of interest may be "moved" between
chromosomes of different parental origin. This is of value when
positional effects influence expression of the nucleotide sequence
of interest.
[0088] Alternatively, the targeting cassette may be introduced into
the plant genome using transformation techniques known in the art.
Thus, tissue obtained from a plant comprising the target site
integrated within its genome, preferably within a recipient
chromosome, may be transformed with a DNA construct comprising the
targeting cassette. Again, in the presence of a compatible
recombinase, the nucleotide sequence of interest flanked by the
non-identical sites of the targeting cassette is integrated into
the target site residing on the chromosome of interest, preferably
a recipient chromosome.
[0089] It is recognized that the compatible recombinase can be
provided by any means known in the art. That is, it can be provided
in a plant cell by transforming the plant with an expression
cassette capable of expressing the recombinase in the plant, by
transient expression, or by providing messenger RNA (mRNA) for the
recombinase or the recombinase protein.
[0090] In one embodiment, hybrid tissue obtained from an F1 hybrid
plant or seed of the invention is cotransformed with a DNA
construct comprising the targeting cassette and with a DNA
construct comprising the compatible recombinase gene operably
linked to an inducible promoter. Use of an inducible promoter
allows for tight regulation of recombinase expression such that
insertion of the nucleotide sequences of interest into the target
site is predictable and stably maintained. In this manner, the
recombinase catalyzes insertion of the nucleotide sequences of
interest into the target site in response to a stimulus, such as a
chemical or environmental stimulus.
[0091] As noted above, the non-identical recombination sites in the
targeting cassette correspond to those in the target site of the
chromosome of interest. That is, if the target site of the
chromosome of interest contains flanking non-identical
recombination sites of FRT and a mutant FRT, the targeting cassette
will contain the same FRT and mutant FRT non-identical
recombination sites.
[0092] It is furthermore recognized that the particular recombinase
used in the invention will depend upon the non-identical
recombination sites in the target site of the chromosome of
interest and the targeting cassette. That is, if FRT sites are
utilized, the FLP recombinase will be needed. In the same manner,
where lox sites are utilized, the Cre recombinase is required. If
the non-identical recombination sites comprise both an FRT and a
lox site, both the FLP and Cre recombinase will be required in the
plant cell to bring about the recombination event.
[0093] The FLP recombinase is a protein that catalyzes a
site-specific reaction that is involved in amplifying the copy
number of the 2-.mu. plasmid of Saccharomyces cerevisiae during DNA
replication. FLP protein has been cloned and expressed. See, for
example, Cox (1993) Proc. Natl. Acad. Sci. USA 80:4223-4227, herein
incorporated by reference. The FLP recombinase for use in the
invention may be that derived from the genus Saccharomyces. It may
be preferable to synthesize the recombinase using plant-preferred
codons for optimum expression in a plant of interest. The
bacteriophage recombinase Cre catalyzes site-specific recombination
between two lox sites. The Cre recombinase is known in the art.
See, for example, Guo et al. (1997) Nature 389:40-46; Abremski et
al. (1984) J. Biol. Chem. 259:1509-1514; Chen et al. (1996) Somat.
Cell Mol. Genet. 22:477-488; and Shaikh et al. (1977) J. Biol.
Chem. 272:5695-5702; herein incorporated by reference. The Cre
recombinase may also be synthesized using plant-preferred
codons.
[0094] It is recognized that many variations of the targeted
insertion aspect of the invention can be practiced. See for example
WO9925821; WO9925855; WO9925840; herein incorporated by
reference.
[0095] Thus the methods of the invention provide a means for
transformation of recipient plants with nucleotide sequences of
interest, regeneration of transgenic recipient plants comprising
nucleotide sequences of interest, and targeted integration of
nucleotide sequences of interest into a chromosome derived from a
recipient plant. The methods are particularly useful for obtaining
transgenic recalcitrant inbreds having the nucleotide sequences of
interest integrated within a chromosome derived from a recalcitrant
inbred.
[0096] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0097] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
Example 1
Embryogenic Response and Transformability of Hi-II X Elite Hybrid
Immature Embryos
[0098] Crosses were made between Hi-II, a highly transformable
genotype, and several recalcitrant elite inbred lines, to generate
hybrid immature embryos. Hybrid immature embryos were collected
approximately 10 DAP and utilized immediately for transformation
using Agrobacterium as a transformation vector.
Agrobacterium-mediated transformation protocol for Hi-II is as
follows. All culture media are presented in the Appendix.
[0099] Preparation of Agrobacterium
[0100] The engineered Agrobacterium tumefaciens LBA4404 was
constructed as per U.S. Pat. No. 5,591,616 to contain a gene of
interest and a selectable marker gene, typically BAR (D'Halluin et
al. (1992) Methods Enzymol. 216:415-426) or PAT (Wohlleben et al.
(1988) Gene 70:25-37).
[0101] To use the engineered vector in plant transformation, a
master plate of single bacterial colonies was first prepared by
inoculating the bacteria on minimal AB medium and then incubating
the bacteria plate inverted at 28.degree. C. in darkness for about
3 days. A working plate was then prepared by selecting a single
colony from the plate of minimal A medium and streaking it across a
plate of YP medium. The YP-medium bacterial plate was then
incubated inverted at 28.degree. C. in darkness for 1-2 days.
[0102] Agrobacterium for plant transfection and co-cultivation was
prepared 1 day prior to transformation. About 30 ml of minimal A
medium in a flask containing 50 .mu.g/ml spectinomycin was
inoculated with a 1/8 loopful of Agrobacterium from a 1- 2-day-old
working plate. The Agrobacterium was then grown at 28.degree. C. at
200 rpm in darkness overnight (about 14 hours). In mid-log phase,
the Agrobacterium was harvested and resuspended at 3 to
5.times.10.sup.8 CFU/ml in 561Q medium+100 .mu.M acetosyringone
using standard microbial techniques and standard curves.
[0103] Immature Embryo Preparation
[0104] Nine to ten days after controlled pollination of a corn
plant, developing immature embryos are opaque and 1-1.5 mm long and
are the appropriate size for Agro-infection. The husked ears were
sterilized in 50% commercial bleach and 1 drop Tween for 30
minutes, and then rinsed twice with sterile water. The immature
embryos were aseptically removed from the caryopsis and placed into
2 ml of sterile holding solution comprised of 561Q+100 .mu.M
acetosyringone.
[0105] Agrobacterium Infection and Co-cultivation of Embryos
[0106] Holding solution was decanted from excised immature embryos
and replaced with prepared Agrobacterium. Following gentle mixing
and incubation for about 5 minutes, the Agrobacterium was decanted
from the immature embryos. Immature embryos were then moved to a
plate of 562P medium, scutellum surface upwards, and incubated at
20.degree. C. for 3 days in darkness followed by incubation at
28.degree. C. for 3 days in darkness (see U.S. Pat. No.
5,981,840).
[0107] Selection of Transgenic Events
[0108] Following incubation, the immature embryos were transferred
to 563O medium for selection of events. The transforming DNA
possesses a herbicide-resistance gene, for example the BAR gene,
which confers resistance to bialaphos. At 10- to 14-day intervals,
embryos were transferred to 563O medium. Actively growing putative
transgenic embryogenic tissue was visible in 6-8 weeks.
[0109] Regeneration of T.sub.0 Plants
[0110] Transgenic embryogenic tissue was transferred to 288W medium
and incubated at 28.degree. C. in darkness until somatic embryos
matured, or about 10 to 18 days. Individual matured somatic embryos
with well-defined scutellum and coleoptile were transferred to 272
embryo germination medium and incubated at 28.degree. C. in the
light. After shoots and roots emerged, individual plants were
potted in soil and hardened-off using typical horticultural
methods.
[0111] Confirmation of Transformation
[0112] Putative transgenic events were subjected to analysis to
confirm their transgenic nature. The specific analytical test
performed on any transgenic was dependent on the transgene.
[0113] For example, almost all events were tested for the presence
of the gene of interest by PCR amplification. In those events
produced with A B.T. gene, an ELISA was performed on leaf tissue
from the regenerated plants. And in those events produced with the
GUS gene, tissues were stained with GUS histochemical reagent.
Additionally, T.sub.0 plants were painted with bialaphos herbicide
(1% v/v Liberty.TM.). The subsequent lack of a herbicide-injury
lesion indicated the presence and action of the BAR/PAT transgene,
which conditions for herbicide resistance.
[0114] Results
[0115] Hybrid immature embryos derived from crosses between Hi-II X
proprietary elites can be cultured successfully and transformed
using a Hi-II/Agrobacterium protocol in a genotype-independent
fashion.
[0116] Culture Responses of Hybrid Immature Embryos
[0117] A high frequency and vigorous embryogenic response was
achieved from hybrid embryos of Hi-II and the proprietary
recalcitrant elite inbreds tested when cultured on a medium
typically used for Hi-II (288) (Table 1). The frequency of
embryogenic response was generally greater when Hi-II was the
female. The preponderance of type I or type II response varied
between genotypes and among embryos within a genotype, but immature
embryos across all genotypes possessed sectors of both tissue
phenotypes.
[0118] Embryogenic cultures of hybrid immature embryos were
serially propagated, and were visually selected to propagate with a
friable phenotype. Plants were regenerated from these embryogenic
cultures and produced progeny seed.
2TABLE 1 CULTURE RESPONSE OF HYBRID EMBRYOS (% OF EMBRYOS PRODUCING
EMBRYOGENIC TISSUE) ELITE Cross GENOTYPE X Hi-II Hi-II X
REGENERABLE PHTE4 70 82 Y PHP18 54 65 Y PH05F 71 90 Y PH09B NT* 81
Y PHJ90 59 78 Y PH24E 84 38 Y PHN46 43 54 Y PH21T 77 43 Y PHAA0 NT*
78 Y ASKC27 NT* 92 Y *NT = not tested
[0119] Transformation of Hybrid Immature Embryos
[0120] Transgenic events were produced from hybrid immature embryos
of Hi-II X PHN46 and reciprocals, and Hi-II X PHP 18 and
reciprocals utilizing the Agrobacterium transformation vector and
protocol described above. The frequency of transformation was about
double when Hi-II was the female, in contrast to the reciprocal, in
both PHN46 and PHP 18 combinations (Table 2). Transformation
frequency was measured as a function of the number of immature
embryos that produced a confirmed transgenic event. Considered
individually (not as hybrids), PHN46 and PHP 18 display
transformation frequencies of about 0.5% (throughput of about 70).
Throughput is measured as independent events/year/person that can
be produced. The optimal transformation protocol for the two elites
is not the same as that utilized for Hi-II. In this example,
throughput for the hybrid immature embryos is increased many
fold.
3TABLE 2 TRANSFORMATION OF Hi-II X ELITE HYBRID IMMATURE EMBRYOS
TRANSFORMATION FREQUENCY ELITE GENOTYPE and (throughput) TRANSGENE
X Hi-II Hi-II X PHP18/ubi::B.T. GENE 4.2 9.4 (540) (1204)
PHN46/glb1::milps::glb1 7.9 12.4 (1020) (1593)
[0121] The distribution of event phenotype, type I versus type II,
revealed that type II events occurred with a greater frequency when
Hi-II was the female (Table 3).
4TABLE 3 DISTRIBUTION OF EVENT PHENOTYPE COUNT OF EVENT PHENOTYPES
(frequency) PEDIGREE I II Hi-II X PHP18/ubi::B.T. GENE 8 (0.25) 24
(0.75) PHP18 X Hi-II/ubi::B.T. GENE 2 (0.50) 2 (0.50) Hi-II X 25
(0.50) 25 (0.50) PHN46/glb1::milps::glb1 PHN46 X Hi- 18 (0.78) 5
(0.22) II/glb1::milps::glb1
Example 2
Agrobacterium-mediated Transformation of Hi-II (Female) X Elite
(Male) Hybrid Immature Embryos
[0122] Transformation of a broad range of proprietary elite
genotypes was pursued with Hi-II as the female only. Transgenic
events were produced from hybrid immature embryos of Hi-II X
proprietary elite inbred crosses utilizing the standard
Hi-II/Agrobacterium transformation protocol described in Example 1.
In most all cases, confirmed transgenic events were recovered from
the hybrids at a frequency and throughput significantly greater
than for the comparable inbreds alone (Table 4).
5TABLE 4 TRANSFORMATION OF Hi-II X ELITE HYBRID IMMATURE EMBRYOS
HYBRID TRANSFORMATION THROUGHPUT EFFICIENCY (%) (AND Hi-II X ELITE/
TRANSFORMATION (independent THROUGHPUT) OF ELITE TRANSFORMATION
VECTOR EFFICIENCY (%) events/year/person) PARENT ALONE
PHT05/ubi:ubiint::GUS::pi- nII 39.9 5109 0 (0)
PH21T/ubi:ubiint::GUS::pinII 19.2 2464 ?
PHP02/ubi:ubiint::GUS::pinII 17.6 2255 0.14 (18)
ASKC27/glb1::milps::glb1 9.1 1165 ? PH24E/ubi:ubiint::GUS::pinII
9.0 1165 0 (0) PH05F/ubi:ubiint::GUS::pinII 8.0 1032 ?
PHP18/ubi:ubiint::GUS::pinII 5.4 694 0.55 (70)
PHN46/ubi:ubiint::GUS::pinII 2.4 307 0.57 (73)
PHN46/glb1::milps::glb1 12.0 1700 PHN46/glb1::AGP2tr::glb1 18.0
2600 PHN46/gz::Ht12ss:BHL3n::gz 6.0 870
PH09B/ubi:ubiint::GUS::pinII 0.27 34 0.61 (78)
PH09B/ubi:ubiint::GUS::pinII 42.3 6000 PHAA0/ubi:ubiint::GUS::pinI-
I 0.28 36 0.13 (16) Hi-II control 23.5 3032 NA ? = Unknown or
untested NA = not applicable
Example 3
Culture Response and Transformability of Hybrid Immature Embryos
Obtained from Crosses between Several Transformable and
Recalcitrant Elite Inbred Genotypes
[0123] Crosses were made between Hi-II or A188, another
transformable maize genotype, and various elite inbred lines such
as Dabo-1, Dabo-2, and Dabo-12. Culture response and
transformability using particle-gun transformation or
Agrobacterium-mediated transformation were examined in inbred
immature embryos and hybrid immature embryos obtained from crosses
between inbreds and elite inbreds. Agrobacterium-mediated
transformation was performed as in Example 1. Particle-gun
transformation protocol was as follows.
[0124] Preparation of Target Tissue
[0125] Immature maize embryos were isolated from ears 9-11 days
after pollination using a scalpel. Prior to isolation the ears were
surface sterilized in 30% Chlorox bleach plus 0.5% Micro detergent
for 20 minutes, and rinsed two times with sterile water. The
immature embryos were excised and placed embryo axis side down
(scutellum side up), 25 embryos per plate, on 560Y medium for 4
hours and then aligned within the 2.5-cm target zone in preparation
for bombardment.
[0126] Preparation of DNA
[0127] Plasmid vectors containing Ubi:ubi int:Gus:PinII or
35S:Bar:pinII were made. This Gus plasmid DNA plus plasmid DNA
containing a Bar selectable marker were precipitated onto 1.1 .mu.m
(average diameter) tungsten pellets using a CaCl.sub.2
precipitation procedure as follows:
[0128] 100 .mu.l prepared tungsten particles in water 10 .mu.l (1
.mu.g) DNA in TrisEDTA buffer (1 .mu.g total)
[0129] 100 .mu.l 2.5M CaCl.sub.2
[0130] 10 .mu.l 0.1M spermidine
[0131] Each reagent was added sequentially to the tungsten particle
suspension, while maintained on the multitube vortexer. The final
mixture was sonicated briefly and allowed to incubate under
constant vortexing for 10 minutes. After the precipitation period,
the tubes were centrifuged briefly, liquid removed, washed with 500
ml 100% ethanol, and centrifuged for 30 seconds. Again the liquid
was removed, and 105 .mu.l 100% ethanol was added to the final
tungsten particle pellet. For particle gun bombardment, the
tungsten/DNA particles were briefly sonicated and 10 .mu.l spotted
onto the center of each macrocarrier and allowed to dry about 2
minutes before bombardment.
[0132] Particle Gun Treatment
[0133] The sample plates were bombarded at level #3 in particle gun
#HE34-1 or #HE34-2. All samples received a single shot at 650 PSI,
with a total of ten aliquots taken from each tube of prepared
particles/DNA.
[0134] Subsequent Treatment
[0135] Following bombardment, the embryos were kept on 560Y medium
for 2 days, and then examined for transient GUS expression or
transferred to 560R selection medium containing 3 mg/liter
Bialaphos, and subcultured every 2 weeks. After approximately 10
weeks of selection, selection-resistant callus clones were
transferred to 288J medium to initiate plant regeneration.
Following somatic embryo maturation (2-4 weeks), well-developed
somatic embryos were transferred to medium for germination and
transferred to the lighted culture room. Approximately 7-10 days
later, developing plantlets were transferred to 272V hormone-free
medium in tubes for 7-10 days until plantlets were well
established. Plants are then transferred to inserts in flats
(equivalent to 2.5" pot) containing potting soil and grown for 1
week in a growth chamber, subsequently grown an additional 1-2
weeks in the greenhouse, then transferred to classic 600 pots (1.6
gallon) and grown to maturity. Transformed plant tissues were
assessed for stable expression of GUS.
[0136] Results
[0137] Callus was examined for type I, type II, or mixed type
I-type II phenotype. As seen in Table 5, the inbred A188 UM showed
the highest percentage of type II phenotype, the inbred AP84 showed
the highest percentage of type I phenotype, and the inbreds Dabo-I
and Dabo-12 showed the highest percentage of mixed type I-type II
phenotype. Among the hybrids, Hi-II showed the highest percentage
of type II phenotype, while the hybrid cross AP84 X PHN46 showed
the highest percentage of type I phenotype.
6TABLE 5 CALLUS PHENOTYPE OF SEVERAL MAIZE INBREDS AND HYBRIDS
(TYPE I/TYPE II/MIXED TYPE I AND TYPE II) Female Selves PHN46 AP84
A188 A188 68.5/0/23 72.5/0/0 85/8.4/0 A188 UM 0/100/0 47.5/0/5
46/38/6 A188 IFS 24/12/0 A188 JT 27.5/8/0 93.8/1.25/0 100/0/0 Hi-II
0/89/0 5/91.2/3.8 0/84/0 Dabo-1 17.6/6.8/59.6 90.5/0/0 43/28.1/20.3
Dabo-2 0/87.2/12.3 Dabo-12 23.3/16.7/43.3 100/0/0 AP84 91/0/0
100/0/0
[0138] Transient and stable GUS expression were measured as
described elsewhere (see Jefferson (1987) Plant Mol Biol. Rep.
5:387-405) following staining of embryo or plant tissue with X-Gluc
staining solution (McCabe et al. (1988) Bio/Technology
6(87):923-926) for 12 h at 37.degree. C. in the dark. Transient and
stable expression data are shown in Tables 6 and 7,
respectively.
7TABLE 6 TRANSIENT GUS EXPRESSION (%) FROM AGROBACTERIUM- MEDIATED
AND PARTICLE GUN TRANSFORMATION EVENTS AND WITH DIFFERENT MAIZE
GENOTYPES Selves PHN46 AP84 A188 Female (gun/Agro) (gun/Agro)
(gun/Agro) (gun/Agro) A188 7.5/25 1/0 60/19.50 A188 UM 50/20 25/0
3/40 A188 IFS 35/35 A188 JT 4.75/55 34/2 62.5/42 Hi-II 5.25/65
21/17.50 22/13 Dabo-1 43.25/16.5 32.5/8 43/12 Dabo-2 32.75/11
Dabo-12 28.5/10.5 75/4.5 AP84 2/13.25 85/1.5
[0139]
8TABLE 7 STABLE GUS EXPRESSION (%) FROM PARTICLE GUN (Gun) AND
AGROBACTERIUM-MEDIATED (Agro) TRANSFORMATION EVENTS AND WITH
DIFFERENT MAIZE GENOTYPES Selves PHN46 AP84 A188 Female (Gun %/Agro
%) (Gun %/Agro %) (Gun %/Agro %) (Gun %/Agro %) A188 4.25/0 4.3/0
7.4/1.05 A188 UM 4.9/0 2.3/3.2 0/4.55 A188 IFS 3.15/0 A188 JT 3.4/0
12.7/5 8.7/2.85 Hi-II 0/1 2.05/0 10/0 Dabo-1 4.12/0 0/0 4.7/1.7
Dabo-2 0/0 Dabo-12 0/0 5.3/0 AP84 0/1 7.5/0
[0140] These data indicate that A188 inbreds and Dabo inbreds
showed similar transformation efficiency of about 4% with
particle-gun transformation. Of the hybrid crosses, A188JT X HG11
and A188JT X AP84 showed the highest transformation efficiency with
both particle gun and Agrobacterium-mediated transformation. The
best overall hybrid genotypes of those tested for transformation
efficiency were A188JT X PHN46 (12.7%) for particle gun
transformation, and Al 88JT X PHN46 (5.0%) and A188UM X AP84
(4.55%) for Agrobacterium-mediated transformation.
Example 4
Identification of Ancestral Origin of Transgenic Chromosomes
Transgenic Materials
[0141] Two types of transgenic materials were used in the present
study to test whether or not flanking sequences of a transgene can
be reliably used to identify parental origins of transgenic
chromosomes in the F1 hybrid transformation system.
[0142] The first type of material was generated by co-bombardment
of immature maize embryos of the genotype Hi-II with two plasmids,
one carrying a gene of interest, hordothionin (Ht12) (U.S. Pat.
Nos. 5,885,801 and 5,885,802 issued Mar. 23, 1999 and WO9940209;
all incorporated by reference herewithin) and the other carrying a
selectable marker gene, pat or bar. Hordothionin is a synthetic
gene that encodes the hordothionin protein containing a high level
of lysine and thus has nutrition value in the feed industry.
Regenerated transformed (T.sub.0) plants from the transformed Hi-II
embryos were crossed to a Pioneer elite inbred line, PHN46, and the
resulting transgenic F1 hybrid progeny (Hi-II X PHN46) were used in
this study. Since the Ht12 genes in those events were originally
transformed into Hi-II chromosomes, we can use these materials to
demonstrate whether or not determination of parental origins of
transgenic chromosomes via genomic sequences flanking transgenes is
affected by the presence of chromosomes from other parental lines
in future crossing generations.
[0143] The other type of material used in the present study was
generated by transformation with the milps gene along with a
selectable marker gene, pat, through Agrobacterium-mediated
transformation (PHP6). Milps is one of the genes involved in the
biochemical pathways of inositol phosphate synthesis (see
WO99/05298, incorporated by reference). The gene was introduced
into immature F1 hybrid maize embryos obtained from the cross of
Hi-II with the elite inbred PHN46 (Hi-II X PHN46) and the resulting
transformed (T.sub.0) plants were used in this study. Since the
transgenes were directly transformed into F1 hybrid chromosomes, it
was unknown into which parental chromosome the transgene was
integrated in these events.
[0144] Plant DNA Extraction and Southern Hybridization
[0145] Isolation of plant genomic DNA, enzyme restriction
digestion, agarose gel electrophoresis, Southern blotting and
hybridization were performed according to Zhong et al. (1999)
"Commercial production of aprotinin in transgenic maize seeds,"
Mol. Breed.: 5:345-356.
[0146] Cloning Genomic Flanking Sequences of a Transgene
[0147] 1. Plasmid Rescue
[0148] In the present study, genomic sequences flanking transgenic
Ht12 DNAs from the events TC1 (co-transformed with plasmids PHP9
and PHP7) and TC0 (co-transformed with plasmids PHP1 and PHP8) were
isolated through the plasmid-rescue approach (Behringer and Medford
(1992), and Feldmann (1992) supra).
[0149] TC1 and TC0 DNA was digested with the restriction enzymes
NsiI and EcoRI, respectively, at 37.degree. C. for 6 hrs.
Restriction enzymes were chosen for digestion in such a way that
the length of fragments released from integrated plasmid DNA was
technically desirable for self-ligation and the origin of
replication and amp gene from the plasmid backbones were kept
intact. The digestion products were circularized by self-ligation
according to the procedures described in the Clontech manufacture
user manual (Clontech Laboratories, Inc., Palo Alto, Calif.). The
circular DNA molecules were then transformed into E. coli following
the conditions of the Gibco BRL manufacture manual and the
transformed cells were selected on bacterial medium with 70-100 ng
/.mu.l ampicillin.
[0150] Those that survived over ampicillin selection on medium were
further amplified and subjected to restriction and sequencing
analyses to determine whether or not the selected clones carried a
host DNA sequence adjunct to transgenic DNA.
[0151] 2. Inverse PCR
[0152] Sequences flanking transgenic DNA from transgenic milps
events were obtained by using an inverse-PCR technique (Gasch et
al. (1992), Britt and Earp (1994), supra).
[0153] DNA from transgenic plants was digested with the restriction
enzyme NheI, which had only one restriction site in the T-DNA. The
digestion products were circularized by self-ligation following the
same ligation procedures described above for plasmid rescue. The
circularized DNAs were amplified with a pair of divergent
transgene-based primers and the products from the first round of
PCR were further amplified with a second set of primers that were
nested within the first set of primers. Amplified PCR products were
subjected to restriction and sequencing analyses to determine
whether or not they contained a host-plant DNA sequence.
[0154] Identification of Transgenic Chromosomes in F1 Hybrids
[0155] When there is a parental restriction fragment length (RFLP)
or single nucleotide polymorphism (SNP) in the genomic regions
closely flanking a transgene, it is possible to use the
polymorphism information to determine which parental chromosome(s)
the transgene is integrated into.
[0156] RFLP analysis followed the Southern protocols described
earlier. The genomic sequences flanking transgenic DNA were used as
probes to reveal the restriction patterns of both parents and
transgenic hybrids. If an RFLP marker(s) that is present in one of
the two parents, parent A for example, is replaced by a new RFLP
marker(s) in their transgenic hybrid, the transgene must be located
on a chromosome from the parent A.
[0157] SNP analysis was accomplished by comparing the profiles of
flanking sequences from both parents and their transgenic hybrids.
Sequence profiles from transgenic hybrids should match that from
one of the parents, by which a parental transgenic chromosome can
be determined. Sequence analysis was carried out on an ABI Prism
377 sequencer manufactured by Perkin Elemer.
[0158] Verification of the flanking sequence in identification of
chromosome identity of parent
[0159] 1. Cloning Genomic Sequences Flanking Transgenic DNA in TC1
and TC0
[0160] Genomic sequences flanking transgenic Ht12 DNA were isolated
from the events TC1 and TC0 by using a plasmid-rescue approach. As
explained earlier, the events were generated by particle
bombardment in Hi-II background and crossed to a Pioneer inbred
line PHN46. Several self-ligated clones from the NsiI or EcoRI
digested transgenic DNA survived over antibiotic selection from
both events. Two clones, designated as 19-2 and 28, were
respectively obtained from TC1 and TC0 and were selected for
further analysis. The clones 19-2 and 28 were about 5 Kb and 3.2 Kb
long, respectively. Restriction enzyme analysis confirmed that the
original cloning sites were retained in the clones. Further
analysis revealed that at least one restriction site that was not
in either of the two co-transformed plasmids was present in the
clones (FIG. 1). This strongly suggested the existence of a stretch
of non-plasmid DNA sequence in the clones selected.
[0161] 2. Determination of the Parental Origins of the Transgenic
Chromosomes in TC1 and TC0 events
[0162] To verify that the Ht12 was originally transformed into a
Hi-II chromosome in TC1, RFLP profiles of Hi-II, PHN46, and TC1
were compared (FIG. 2). DNA samples from those lines were digested
with the restriction enzyme BamHI and probed with a 700 bp
non-plasmid DNA fragment isolated from the clone 19-2. Hi-II and
PHN46 each showed a single hybridized band but the Hi-II's
hybridized fragment (about 7 kb) was much larger than that of PHN46
(about 1.0 kb). Two hybridization fragments were observed in TC1.
One of them was equivalent in size to that of PHN46 while the other
was smaller than that of Hi-II (6.5 kb vs. 7 kb). No corresponding
Hi-II fragment was observed in TC1. These results suggested that
the Ht12 gene in TC1 must have originally integrated into a Hi-II
chromosome, which agreed with the known fact described above.
[0163] The transgenic DNA on a Hi-II chromosome in TC0 was
confirmed through SNP analysis. The SNP profiles in the genomic
regions flanking the transgenic DNA among Hi-II, PHN46, and TC0 are
presented in FIG. 3. A total of 6 SNPs were observed between Hi-II
and PHN46 in the amplified PCR products. The sequencing profile of
TC0 was the same as that of Hi-II, confirming that the transgenic
DNA was integrated into a Hi-II chromosome in TC0.
[0164] Identification of the Parental Origins of Transgenic
Chromosomes in Hi-IIxPHN46 F1 Hybrids
[0165] 1. Cloning Genomic Sequences Flanking Transgenes in
Transgenic Hi-II X PHN46 F1 Hybrids
[0166] Inverse-PCR technique was used to clone genomic sequences
flanking transgenic milps expression cassettes in Hi-II X PHN46 F1
hybrids. Sequences contiguous to the right borders of T-DNA were
obtained from more than 10 milps events. In most cases, a second
PCR was necessary to obtain specific amplification of a desired PCR
product (FIG. 4). The length of flanking sequences cloned from the
study varied, ranging from below 200 bp up to more than 1 kb
long.
[0167] 2. Determination of the Parental Origins of Transgenic
Chromosomes in Milps Events
[0168] Parental origins of transgenic chromosomes in 6 milps events
(Table 8) were determined, two by Southern blot and four by SNP
analyses, in this study. FIG. 5 shows the hybridization patterns of
Hi-II, PHN46, and the F1 transgenic milps event 2482.53-1-12A. The
DNA samples were digested by the restriction enzyme NheI and the
blot was probed with a flanking sequence isolated from the
transgenic event 2482.53-1-12A. Hi-II showed two hybridized bands
(1.4 and 0.6 kbs) that were also present in the F1 transgenic
hybrid 2482.53-1-12A. Three hybridization bands were observed in
PHN46 and one of them (0.6 kb) was also present in both Hi-II and
2482.53-1-12A. The two other PHN46 bands (1.5 and 1.1 kbs) were not
present in 2482.53-1-12A. Instead, they were replaced by a novel
band (6.0 kb) in 2482.53-1-12A. These results suggest that the
milps gene in 2482.53-1-12A integrated into a PHN46 chromosome.
9TABLE 8 CLONED GENOMIC SEQUENCES FLANKING MI1PS EXPRESSION
CASSETTES AND THEIR USES IN IDENTIFYING THE PARENTAL ORIGINS OF
TRANSGENIC CHROMOSOMES IN SIX TRANSGENIC F1 HYBRID EVENTS Parental
Origin of Trans- genic Gene of Transformation Transformation
Chromo- Event Code Interest Method Target somes 2482.53-1-1 MI1PS
Agrobacterium F1, Hi-IIxPHN46 PHN46 2482.53-1-8 MI1PS Agrobacterium
F1, Hi-IIxPHN46 Hi-II 2482.53-1-12A MI1PS Agrobacterium F1,
Hi-IIxPHN46 PHN46 2482.53-1-12B MI1PS Agrobacterium F1, Hi-IIxPHN46
PHN4 2482.53-1-3 MI1PS Agrobacterium F1, Hi-IIxPHN46 Hi-II
2482.53-1-5 MI1PS Agrobacterium F1, Hi-IIxPHN46 Hi-II
[0169] An example of determining the parental origins of transgenic
chromosomes in F1 transgenic milps events through SNP analysis is
shown in FIG. 6. In this example, a 684 bp-long genomic sequence
(designated as JR451) contiguous to the right border of the
transgenic DNA in the event 2482.53-1-12B was cloned. Sequences in
the corresponding genomic regions were amplified from Hi-II, PHN46,
and the transgenic event 2482.53-1-12B. A total of 27 SNPs were
observed between Hi-II and PHN46 in the amplified PCR products. The
sequencing profile of 2482.53-1-12B was the same as that of PHN46,
suggesting that the trangenic DNA must have integrated into a PHN46
chromosome in 2482.53-1-12B.
Example 5
Biolistics Transformation of Immature Embryos of Hi-II x PHN46, a
Proprietary Elite Inbred
[0170] Preparation of Target Tissue:
[0171] Ears from reciprocal crosses of Hi-II and PHN46 were
produced from greenhouse or field grown plants. Ears were harvested
based on developmental stage of the immature embryo and used when
the embryo becomes opaque about 8-13 DAP, depending on genotype and
environmental conditions.
[0172] The ears were surface sterilized in 50% Clorox bleach+0.5%
Micro detergent for 20 minutes, and rinsed 2X with sterile water.
Immature embryos were aseptically dissected from sterilized ears
and placed embryo axis side down (scutellum side up) on 560L in
Petri dishes and cultured in darkness at 28.degree. C. After 4-5
days the embryos were transferred to 560Y for 4 hours, arranged
within the 2 cm target zone at 10 embryos per plate. The embryos
were oriented with the coleorhizal end pointing up at approximately
a 30.degree. angle.
[0173] Particle Gun Bombardments:
[0174] Particles and DNA were associated as described below in
Particle-DNA Association. The target plates were bombarded at shelf
2 (8.2 cm from rupture disk) in PDS-1000 following the
manufacturers recommendations. Immature embryos received a single
bombardment at 650 PSI at 28.degree. C. in Hg vacuum.
[0175] Selection of Transformants:
[0176] Following bombardment, the embryos remained on 560Y for 2
days in darkness at 28.degree. C. The embryos were then transferred
to 560R selection medium containing 3 mg/liter bialaphos and
cultured in darkness at 28.degree. C. The embryos were transferred
to fresh medium of the same composition every 10-14 days.
[0177] Six to twelve weeks following bombardment,
bialaphos-resistant embryogenic tissue were produced from the
immature embryos. The tissues from individual embryos were
identified as putative transgenic events and were individually
subcultured and propagated on 560R. Analysis to document the
transgenic nature of the herbicide-resistant events was carried out
at this stage.
[0178] Fragments of embryogenic tissue from events chosen for
regeneration were subcultured 288J maturation medium in
100.times.25 plates. The tissue was grown in darkness at 28.degree.
C. After about 10-14 days, matured somatic embryos were
individually transferred to 272V, germination medium, in
100.times.25 Petri plates and maintained at 28.degree. C. in light.
Approximately 7-10 days later, developing plants were transferred
to 272V medium in 150.times.25 mm culture tubes and incubated for
7-10 days until roots were well-formed and new aerial growth is
well-established.
[0179] Plants were then transferred to inserts in flats (equivalent
to 2.5"pot) containing potting soil and grown for 1 week in a
humidified growth chamber. The flats of regenerated plants were
grown an additional 1-2 weeks in the greenhouse for hardening-off.
Finally, individual regenerated plants were transplanted to 1.6
gallon pots and grown to maturity.
[0180] Particle Preparation and Particle-DNA Association
[0181] Preparation of Tungsten Particles:
[0182] 1) Weigh 60 mg 1u GE W particles into 15 ml centrifuge
tube.
[0183] 2) Add 2 ml 0.1M HNO.sub.3 and sonicate on ice for 20
minutes.
[0184] 3) Withdraw acid, add 1 ml sterile deionized water and
transfer sample to a 2 ml Sarstedt tube. Sonicate briefly.
[0185] 4) Centrifuge to pellet particles
[0186] 5) Withdraw water and add 1 ml 100% EtOH. Sonicate
briefly.
[0187] 6) Centrifuge to pellet particles
[0188] 7) Withdraw EtOH and add 1 ml 100% EtOH. Sonicate
briefly.
[0189] 8) Centrifuge to pellet particles
[0190] 9) Withdraw EtOH and add 1 ml sterile deionized water.
Sonicate. Pipet 250 ml of suspension into 4, 2 ml tubes. Add 750 ml
of sterile deionized water to each tube. Freeze tungsten sample
between uses.
[0191] Particle/DNA Association--CaC12/Spermidine Method:
[0192] 1) 100 ul prepared 1u W particles dispensed into siliconized
tube
[0193] 2) 10 ul (1 ug total) plasmid DNA in TE buffer
[0194] 3) 100 ul 2.5 M CaCl.sub.2
[0195] 4) 10 ul 0.1 M spermidine
[0196] Each reagent is added sequentially to the particle
suspension with gentle vortexing. After addition of all components,
the preparation was vortexed at a setting #3-4 for ten minutes.
After the association period, the tubes were centrifuged briefly,
the supernatant decanted, and the particle-DNA amalgam was washed
with 500 ml 100% ethanol, and the particles were pelleted by
centrifugation for 30 seconds in a microfuge. The supernatant was
removed, and the particle-DNA association is resuspended in 105 ul
100% ethanol. Prior to bombardment, the associated particles-DNA
were briefly sonicated and 10 ul of the suspension was spotted onto
the center of each macro-carrier and allowed to dry for
approximately 2 minutes before bombardment.
[0197] Results:
[0198] Production of Transformants:
[0199] Transgenic events were produced from embryos derived from
reciprocal crosses of Hi-II and PHN46 using construct PHP6A
(gz::HT12::gz)+PHPO (ubi::PATmo::pinII). The events were confirmed
by PCR reaction for the agronomic gene of interest (gz::HT12::gz).
In this comparison, the production of transgenic events via
particle gun is documented, as well as using Hi-II as the female in
the production of the hybrid target embryos.
10 TABLE 9 Transformation Genotype Frequency Hi-II X PHN46 4.5%
PHN46 X Hi-II 2.3%
Example 6
Seed Production from Hybrid Transformants
[0200] Transgenic events were produced from a variety of hybrids of
Hi-II X proprietary elite inbred using proprietary commercially
valuable gene constructs in an Agrobacterium vector. T.sub.0 plants
were regenerated and established in a greenhouse to recover progeny
T.sub.1 seed.
[0201] The performance of T.sub.0 plants from hybrid transformants
was measured by plant survival to reproductive maturity and seed
production. Comparisons were made relative to Hi-II transformants
alone. Comparisons were conducted with T.sub.0's that occupied the
greenhouse contemporaneously. In these comparisons, the survival
and seed production of transgenic events derived from hybrids far
surpassed the performance of Hi-II transformants.
11TABLE 10 Performance of T.sub.0 Plants from Hybrid Transformants
Survival and Average Seed % T.sub.0 Pollinated and Average Seed
T.sub.0 Genotype Harvested per T.sub.0 Hi-II 67 85.4 HYBRID 85
131.4 (pooled) selfed 121.5 crossed 174.3 Performance of T.sub.0
Plants from Hybrid Transformants Distribution of Seed Production
T.sub.0 Genotype T.sub.0 Seed Count Hi-II Hybrid selfed Hybrid
crossed >20 66 74 90 >40 58 65 81 >60 50 61 75 Percentage
of Occurrence
Example 7
Dicots
[0202] Crosses are made between a soybean variety that is a
particular transformable line, such as "Jack", and a recalcitrant,
commercially desirable line such as Pioneer 93B82. F1 embryos of
Jack x 93B82 are transformed using biolistics techniques with
embryogenic cultures derived from immature embryos (see Klein et
al. (1987) Nature (London) 327:70; WO0032782; and WO0028058, which
are incorporated by reference).
[0203] Following bombardment, selection for transformed events is
performed and transformed plant tissues are tested for stable
expression of one or more transgenes. Transgenic tissue is used for
isolation of plant genomic DNA, enzyme restriction digestion,
agarose gel electrophoresis, Southern blotting and hybridization as
described in Zhong et al, supra, and as known in the art.
[0204] Genomic DNA containing flanking sequences of the F1
transformed event and that from one or more parents are isolated
and may be cut with restriction enzymes and/or sequenced.
Transgenic chromosomes are identified as described in previous
examples.
12TABLE 11 561 Q Ingredient Amount Unit D-I Water, Filtered 950.000
ml Chu (N6) Basal Salts (Sigma C-1416) 4.000 g Eriksson's Vitamin
Mix (1000x Sigma-1511) 1.000 ml Thiamine.HCL .4 mg/ml 1.250 ml 2,
4-D 0.5 mg/ml (No. 2A) 3.000 ml L-proline 0.690 g Sucrose 68.500 g
Glucose 36.000 g Directions # = Add after sterilizing Dissolve
ingredients in polished D-I H.sub.2O in sequence Adjust pH to 5.2
w/KOH Q.S. to volume with polished D-I H.sub.2O after adjusting pH
Filter sterilize (do not autoclave)
[0205]
13TABLE 12 562 P Ingredient Amount Unit D-I Water, Filtered 950.000
ml Chu (N6) Basal Salts (Sigma C-1416) 4.000 g Eriksson's Vitamin
Mix (1000x Sigma-1511) 1.000 ml Thiamine.HCL .4 mg/ml 1.250 ml 2,
4-D 0.5 mg/ml 4.000 ml L-proline 0.690 g Sucrose 30.000 g Gelrite @
3.000 g Silver Nitrate 2 mg/ml # 0.425 ml Aceto Syringone 100 mM #
1.000 ml Directions @ = Add after Q.S. to volume # = Add after
sterilizing and cooling to temperature Dissolve ingredients in
polished D-I H.sub.2O in sequence Adjust pH to 5.8 w/KOH Q.S. to
volume with polished D-I H.sub.2O after adjusting pH Sterilize and
cool to 60.degree. C.
[0206]
14TABLE 13 563 O Ingredient Amount Unit D-I Water, Filtered 950.000
ml Chu (N6) Basal Salts (Sigma C-1416) 4.000 g Eriksson's Vitamin
Mix (1000x Sigma-1511) 1.000 ml Thiamine.HCL .4 mg/ml 1.250 ml
Sucrose 30.000 g 2, 4-D 0.5 mg/ml (No. 2A) 3.000 ml L-proline 0.690
g Mes Buffer 0.500 g Agar (Sigma A-7049, Purified) @ 8.000 g Silver
Nitrate 2 mg/ml # 0.425 ml Bialaphos 1 mg/ml # 3.000 ml Agribio
Carbenicillin 50 mg/ml # 2.000 ml Directions @ = Add after Q.S. to
volume # = Add after sterilizing and cooling to temperature
Dissolve ingredients in polished D-I H.sub.2O in sequence Adjust to
pH 5.8 w/koh Q.S. to volume with polished D-I H.sub.2O after
adjusting pH Sterilize and cool to 60.degree. C.
[0207]
15TABLE 14 288 W Ingredient Amount Unit D-I H.sub.2O 950.000 ml MS
Salts 4.300 g Myo-Inositol 0.100 g MS Vitamins Stock Solution (No.
36J) 5.000 ml Zeatin .5 mg/ml 1.000 ml Sucrose 60.000 g Agar (Sigma
A-7049, Purified) @ 8.000 g IAA 0.5 mg/ml # 2.000 ml .1 Mm ABA #
1.000 ml Bialaphos 1 mg/ml # 3.000 ml Agribio Carbenicillin 50
mg/ml # 2.000 ml Directions @ = Add after Q.S. to volume # = Add
after sterilizing and cooling to temperature Dissolve ingredients
in polished D-I H.sub.2O in sequence Adjust to pH 5.6 Q.S. to
volume with polished D-I H.sub.2O after adjusting pH Sterilize and
cool to 60.degree. C. Add 3.5 g/L of Gelrite for cell biology
[0208]
16TABLE 15 272 Ingredient Amount Unit D-I H.sub.2O 950.000 ml MS
Salts 4.300 g Myo-Inositol 0.100 g MS Vitamins Stock Solution 5.000
ml Sucrose 40.000 g Gelrite @ 1.500 g Directions @ = Add after Q.S
to volume Dissolve ingredients in polished D-I H.sub.2O in sequence
Adjust to pH 5.6 Q.S. to volume with polished D-I H.sub.2O after
adjusting pH Sterilize and cool to 60.degree. C.
[0209]
17TABLE 16 272 V Ingredient Amount Unit D-I H.sub.2O 950.000 Ml MS
Salts (GIBCO 11117-074) 4.300 G Myo-Inositol 0.100 G MS Vitamins
Stock Solution ## 5.000 Ml Sucrose 40.000 G Bacto-Agar @ 6.000 G
Directions: @ = Add after bringing up to volume Dissolve
ingredients in polished D-I H.sub.2O in sequence Adjust to pH 5.6
Bring up to volume with polished D-I H.sub.2O after adjusting pH
Sterilize and cool to 60.degree. C. ## = Dissolve 0.100 g of
Nicotinic Acid; 0.020 g of Thiamine.HCL; 0.100 g of Pyridoxine.HCL;
and 0.400 g of Glycine in 875.00 ml of polished D-I H.sub.2O in
sequence. Bring up to volume with polished D-I H.sub.2O. Make in
400 ml portions. Thiamine.HCL & Pyridoxine.HCL are kept in a
dark desiccator. Store for one month, unless contamination or
precipitation occurs, then make fresh stock. Total Volume (L) =
1.00
[0210]
18TABLE 17 288 J Ingredient Amount Unit D-I H.sub.2O 950.000 Ml MS
Salts 4.300 g Myo-Inositol 0.100 g MS Vitamins Stock Solution ##
5.000 ml Zeatin .5 mg/ml 1.000 ml Sucrose 60.000 g Gelrite @ 3.000
g Indoleacetic Acid 0.5 mg/ml # 2.000 ml 0.1 mM Abscisic Acid 1.000
ml Bialaphos 1 mg/ml # 3.000 ml Directions: @ = Add after bringing
up to volume Dissolve ingredients in polished D-I H.sub.2O in
sequence Adjust to pH 5.6 Bring up to volume with polished D-I
H.sub.2O after adjusting pH Sterilize and cool to 60.degree. C. Add
3.5 g/L of Geirite for cell biology. ## = Dissolve 0.100 g of
Nicotinic Acid; 0.020 g of Thiamine.HCL; 0.100 g of Pyridoxine.HCL;
and 0.400 g of Glycine in 875.00 ml of polished D-I H.sub.2O in
sequence. Bring up to volume with polished D-I H.sub.2O. Make in
400 ml portions. Thiamine.HCL & Pyridoxine.HCL are in Dark
Desiccator. Store for one month, unless contamination or
precipitation occurs, then make fresh stock. Total Volume (L) =
1.00
[0211]
19TABLE 18 560 R Ingredient Amount Unit D-I Water, Filtered 950.000
ml CHU (N6) Basal Salts (SIGMA C-1416) 4.000 g Eriksson's Vitamin
Mix (1000X SIGMA-1511 1.000 ml Thiamine.HCL 0.4 mg/ml 1.250 ml
Sucrose 30.000 g 2, 4-D 0.5 mg/ml 4.000 ml Gelrite @ 3.000 g Silver
Nitrate 2 mg/ml # 0.425 ml Bialaphos 1 mg/ml # 3.000 ml Directions:
@ = Add after bringing up to volume # = Add after sterilizing and
cooling to temp. Dissolve ingredients in D-I H.sub.2O in sequence
Adjust to pH 5.8 with KOH Bring up to volume with D-I H.sub.2O
Sterilize and cool to room temp. Total Volume (L) = 1.00
[0212]
20TABLE 19 560 Y Ingredient Amount Unit D-I Water, Filtered 950.000
ml CHU (N6) Basal Salts (SIGMA C-1416) 4.000 g Eriksson's Vitamin
Mix (1000X SIGMA-1511 1.000 ml Thiamine.HCL 0.4 mg/ml 1.250 ml
Sucrose 120.000 g 2, 4-D 0.5 mg/ml 2.000 ml L-Proline 2.880 g
Gelrite @ 2.000 g Silver Nitrate 2 mg/ml # 4.250 ml Directions: @ =
Add after bringing up to volume # = Add after sterilizing and
cooling to temp. Dissolve ingredients in D-I H.sub.2O in sequence
Adjust to pH 5.8 with KOH Bring up to volume with D-I H.sub.2O
Sterilize and cool to room temp. ** Autoclave less time because of
increased sucrose** Total Volume (L) = 1.00
Example 8
Sunflower Hybrids
[0213] When SMF3 was used as a pollen recipient, the plants were
bagged to prevent cross pollination and to isolate the flowers.
This was done by utilizing a finely meshed plastic Delnet bag, and
placing the bag on the developing flower bud at the R4 stage prior
to flowering. To emasculate the plants, since they would be fertile
and pollen producing, at the flowering stage, anthers were removed
by tweezers on a daily basis. After removal, the plants would then
be washed to insure that no pollen persisted on the flowering head.
This process would be done early each morning before pollen would
dehisce from the anthers, to further reduce any chance of selfing.
This emasculation process would go on for several days until all of
the flowers within the composite head would have opened. This
normally takes 5-7 days.
[0214] When commercial sunflower lines were used, the female lines
were made up of both sterile and fertile genotypes. The sterile
lines (F designation) have cytoplasmically controlled sterility
which prevents pollen shedding. The CMS (cytoplasmic male
sterility) was identified and developed by an interspecific cross
or Helianthus annuus and H. petiolaris. The fertile lines consisted
of both maintainers (G designation) and restorers (M designation)
of the CMS genotypes. The same emasculation process was used as
indicated above.
[0215] Hybrids were made by collecting pollen from the SMF3 or from
other genotypes, using small papers. The papers then were used to
cross the pollen on to the genetically or emasculated sterile
plants.
[0216] Sunflower Transformation
[0217] A general method for transformation of sunflower meristem
tissues is practiced as follows (see also European patent number
486233, herein incorporated by reference, and Malone-Schoneberg,
J., et al., Plant Science, 103: 199-207 (1994)).
[0218] Mature sunflower seed (Helianthus annuus L.) of elite line
hybrids or research selection SMF-3 (a selection of USDA germplasm
release SFM-3; cms/H. petiolaris Nuttall//cms HA89 backcross) were
dehulled using a single wheat-head thresher if possible or by hand.
The seed was provided by the Pioneer sunflower research station at
Woodland, Calif. Seeds were surface sterilized for 20 minutes in a
20% Chlorox bleach solution with the addition of two drops of Tween
20 per 50 ml of solution. The seeds were rinsed twice with sterile
distilled water.
[0219] Disarmed Agrobacterium tumefaciens strain EHA105 was used in
all transformation experiments. Binary vector PHP10940 was
introduced into EHA105 using a freeze-thaw transformation method
(Holsters, et al., Mol. Gen. Genet., 163: 181-187 (1978)). The
plasmid contains plant expressed GUS and NPTII genes between the
right and left T-DNA borders. In these experiments we used only the
GUS gene to measure transformation response. Bacteria for plant
transformation experiments were grown overnight (28.degree. C. and
100 RPM continuous agitation) in liquid YEP medium (10 gm/l yeast
extract, 10 gm/l Bactopeptone and 5 gm/l NaCl, pH 7.0) with 50 mg/l
kanamycin. The suspension was used when it reached an OD.sub.600 of
about 0.5 to 1.5. The Agrobacterium cells were pelleted and
re-suspended at a final OD600 of 4.0 in an inoculation medium
comprised of 12.5 mM MES pH 5.7, 1 gm/l NH.sub.4Cl, and 0.3 gm/l
MgSO.sub.4.
[0220] The sunflower transformation protocol allows the recovery of
transgenic progeny without the use of chemical selection pressure,
therefore the NPTII gene in PHP 10940 was not used. Dehulled and
surface-sterilized seeds were imbibed in the dark at 26 C for 20 h
on filter paper moistened with water. The cotyledons and root
radical were removed, and the meristem explants were cultured on
374E (GBA medium consisting of MS salts, Shepard vitamins, 40 mg/l
adenine sulfate, 3 % sucrose, 0.5 mg/l 6-BAP, 0.25 mg/l IAA, 0.1
mg/l GA, and 0.8% Phytagar at pH 5.6) for 24h in the dark. The
primary leaves were removed to expose the apical meristem.
Approximately 40 explants were placed with the apical dome facing
upward in a 2 cm circle in the center of 374M (GBA medium with 1.2%
Phytagar) and then cultured on the medium for 24 h in the dark.
Particle bombardment of the explants with particles containing no
DNA followed this culture step. The particle bombardment is done to
provide improved conditions in the meristem tissue for
Agrobacterium T-DNA transfer.
[0221] Particle bombardment was done by resuspending approximately
18.8 mg of 1.8 .mu.m tungsten particles in 200 .mu.l absolute
ethanol. Particles were sonicated briefly to create a uniform
suspension, then 10 .mu.l of it was dropped on the center of the
surface of macrocarrier. Each plate was bombarded twice using a
BioRad PDS1000/He helium gun with 650 psi rupture discs. The plates
are bombarded on the first shelf of the gun at 26 mm Hg vacuum.
[0222] Particle-bombarded explants were spread out on the 374M
plates and a droplet of Agrobacterium suspension was placed
directly onto the top of each meristem. The explants were
co-cultivated on 374M medium for 4 days then transferred to 374 C
medium (GBA with 1% sucrose and no BAP, IAA, or GA3 and
supplemented with 250 .mu.g/ml cefotaxime). The plantlets were
cultured on 374C medium for about 2 weeks in a culture room set up
for 18h day and a constant temperature of 26 C.
[0223] Sunflower shoots developed in the two-week culture period
following transformation. The shoots were then sacrificed to GUS
staining in order to quantify the transformation response. The
staining was used to identify transformed sectors on T0 shoots. The
GUS staining protocol included McCabe's GUS staining solution.
[0224] Transformation response was quantified by noting both the
frequency of transformed sectors and the quality of the
sectors.
[0225] Transformation Frequency of SMF3 Hybrids with Elite
Sunflower Genetics
[0226] A number of hybrid sunflower lines were developed that
involved crossing commercial line genetics with SMF3. Commercial
lines of sunflower generally do not perform as well as SMF3 in
tissue culture and are also difficult to transform. However, SMF3
is agronomically inferior to commercially available inbreds. This
inferiority leads sunflower breeders to initiate numerous backcross
cycles using commercially viable inbreds as recurrent parents in
order to obtain commercially viable inbreds having the trait
conferred by transformation, but lacking the inferior traits of
SMF3. Experiments were conducted to determine if the transformation
competence of SMF3 could be conferred to lines constituted by
hybridizing SMF3 with commercial inbreds. SMF3 was identified as
the transformation competence donor genotype, while commercial
inbreds constitute the transformation recipient genotypes. The
ability to successfully transform the described hybrids at high
rates would permit breeders to reduce their use of backcrossing.
This would occur since (1) the hybrid would possess 50% genetic
contribution from the elite parent and (2) there would
theoretically be a 50% probability that single transformation
events occurred within the chromosomes donated by the elite parent.
When transformation of the elite chromosome occurs, the
tranformation event has a higher probability of being genetically
linked with favorable traits and this in turn, reduces the need for
backcrossing in order to derive a commercially viable inbred. These
hybrids were developed using SMF3 as both a female (pollen
recipient) and as a male (pollen donor).
[0227] Agrobacterium transformation in these experiments was done
according to the standard protocols used for SMF3. The goal of each
experiment was to do side by side comparisons of the transformation
response of the hybrid and both of the parental lines, SMF3 and a
commercial line. Two weeks after the inoculation with
Agrobacterium, sunflower shoots were removed from tissue culture
and placed in GUS stain. Individual cells and/or sectors of
transformation on the shoots turn blue and were used to quantify
the transformation response. If there are sectors that include
tissues that give rise to germ line cells, then it is possible to
recover the transgene in the next generation of seed. This is the
most desirable result and the lack of this sector type contributes
largely to the difficulty of commercial line transformation.
Quantifying the transformation response included noting how many
sectors have the potential to contribute to the germ line. GUS
staining observed on transformed sunflower was divided roughly into
4 categories (Table 1). The categories include 1) small transformed
cell patches and sectors which most likely do not have the
potential to contribute to the germ line on the lower part of the
shoots; 2) sectors of transformation that most likely would
contribute to the germ line on the lower parts of the shoots; 3)
long sectors that develop to the upper parts of the shoot that
would not contribute to germ line transformation; and 4) larger
sectors that develop to the upper parts of the shoot that are
likely to contribute to germ line transformation. The sector types
that most often result in transgenic seed are those described in
phenotype 2 and 4 (Table 1).
[0228] The transformation response of PR126M alone or in
combination with SMF3 is an example of the increased frequency that
you can obtain with the hybrid combination (Table 2). This
commercial line showed a relatively good transformation response
compared to the other commercial lines that were tested. In the
first experiment (Table 2), PR126M responded as well as the SMF3 X
PR126M hybrid. The enhancement contributed by SMF3, however, was
observed in experiments two and three. A more dramatic difference
is observed using a line with a very poor transformation response
such as VK89M (Table 4). VK89M showed a 10-15% transformation
response. In contrast, the SMF3 X VK89M hybrid exhibited
approximately a three-fold increase in transformability over VK89M.
A similar result occurred when SMF3 was hybridized with PK68G. The
transformation response of the hybrid SMF3 X PK68G was almost
double that of PK68G per se (Table 3). In all of the experiments
listed in Tables 2 -4, SMF3 was used as the female in the
pollinations.
[0229] We also tested the transformation response of lines where
SMF3 was used as the pollen donor (male) in making commercial line
hybrids (Table 5). Experiments conducted with some of these hybrid
lines also showed improved transformation response. The best
examples for improvement are shown by inbreds VK40 and RXT004L
(Table 5). The hybrid combination VK40F X SMF3 showed a 3 fold
increase over inbred line VK40G. A similar increase was also seen
for RXT004LF X SMF3 over RXT004LG.
[0230] Of the fifteen combinations tested, in only one case was the
transformation frequency of the SMF3 hybrid reduced relative to the
commercial inbred transformation recipient (SMF3x VDK612LG) In
contrast, twelve of the fifteen combinations tested showed moderate
to dramatic increases in transformation frequency in the hybrid
relative to the commercial inbred transformation recipient.
21TABLE 20 DESCRIPTION OF CATEGORIES OF GUS PHENOTYPES SCORED IN
HYBRID SUNFLOWER TRANSFORMATION EXPERIMENTS Phenotype Description
Phenotype 1 spots (cells) and streaks (cell files) at the base of
the shoots Phenotype 2 sectors and developing secondary shoots with
sectors at the base of the shoots Phenotype 3 spots (cells) and
streaks (cell files) that develop all the way to the top of the
shoots Phenotype 4 sectors that develop all the way to the top of
the shoot (includes the broad leaf sectors at the tops of
shoots)
[0231]
22TABLE 21 TRANSFORMATION RESPONSE OF PR126M, SMF3, AND SMF3
HYBRIDS WITH PR126M GUS Phen Phen Phen Phen Phen Shoots Pos. 1 2 3
4 2 + Ex. Line (no.) (no.) (no.) (no.) (no.) (no.) 4 (no.) 1 PR126M
70 54 11 8 8 27 35 (77%) (50%) SMF3 68 60 14 20 3 23 43 (88%) (63%)
SMF3 X 70 65 17 15 13 20 35 PR126M (93%) (50%) 2 PR126M 98 79 15 17
23 24 41 (81%) (42%) SMF3 118 109 13 33 21 42 75 (92%) (64%) SMF3 X
82 78 17 12 22 27 39 PR126M (95%) (48%) 3 PR126M 65 55 23 6 16 10
16 (85%) (25%) SMF3 76 73 14 24 9 26 50 (96%) (66%) SMF3 X 56 51 13
8 14 16 24 PR126M (91%) (43%)
[0232]
23TABLE 22 TRANSFORMATION RESPONSE OF PK68G, SMF3, AND SMF3 HYBRIDS
WITH PK68G GUS Phen Phen Phen Phen Phen Shoots Pos. 1 2 3 4 2 + Ex.
Line (no.) (no.) (no.) (no.) (no.) (no.) 4 (no.) 1 PK68G 73 52 19 9
11 13 22 (71%) (30%) SMF3 39 34 6 16 2 10 26 (87%) (67%) SMF3 X 73
67 19 17 13 18 35 PK68G (92%) (48%) 2 PK68G 60 53 23 11 12 7 18
(88%) (30%) SMF3 43 37 8 12 4 13 25 (86%) (58%) SMF3 X 63 54 16 17
8 13 30 PK68G (86%) (48%)
[0233]
24TABLE 23 TRANSFORMATION RESPONSE OF VK89M, SMF3, AND SMF3 HYBRIDS
WITH VK89M GUS Phen Phen Phen Phen Phen Shoots Pos. 1 2 3 4 2 + Ex.
Line (no.) (no.) (no.) (no.) (no.) (no.) 4 (no.) 1 VK89M 84 33 9 4
11 9 13 (39%) (15%) SMF3 87 76 15 21 7 33 54 (87%) (62%) SMF3 X 23
21 6 7 5 3 10 VK89M (91%) (43%) 2 VK89M 86 60 33 5 16 6 11 (70%)
(13%) SMF3 53 48 8 17 6 17 34 (91%) (64%) SMF3 X 80 75 27 18 14 16
34 VK89M (94%) (43%)
[0234]
25TABLE 24 TRANSFORMATION RESPONSE OF COMMERCIAL LINE INBREDS,
SMF3, AND COMMERCIAL LINE HYBRIDS WITH SMF3 GUS Phen Phen Phen Phen
Phen Shoots Pos. 1 2 3 4 2 + Ex. Line (no.) (no.) (no.) (no.) (no.)
(no.) 4 (no.) 1 VK106G 24 14 11 1 0 2 3 (58%) (13%) VK106F 59 54 25
5 13 11 16 X SMF3 (92%) (27%) VK40G 50 35 15 5 11 4 9 (70%) (18%)
VK40F X 59 55 10 16 10 19 35 SMF3 (93%) (59%) SMF3 53 52 7 15 7 23
38 (98%) (72%) 2 SWK- 9 1 0 1 0 0 1 002LG (11%) (11%) SWK- 43 20 10
0 6 4 4 002LF (47%) (9%) X SMF3 RXT004- 34 25 16 0 6 3 3 LG (74%)
(9%) RXT004- 39 30 13 8 6 3 11 LF X (77%) (28%) SMF3 VK820F 18 11 5
0 3 3 3 (61%) (17%) VK820F 44 42 17 7 13 5 12 X SMF3 (95%) (27%)
SMF3 43 42 14 12 7 9 21 (98%) (49%) 3 LC1019G 25 23 11 5 4 3 8
(92%) (32%) LC1019F 47 42 18 6 7 11 17 X SMF3 (89%) (36%) D99G 35
22 6 5 6 5 10 (63%) (21%) D99F X 53 41 17 2 13 9 11 SMF3 (77%)
(29%) VKD612- 54 50 9 16 6 19 35 LG (93%) (65%) VKD612- 45 30 15 6
5 4 10 LF X (67%) (22%) SMF3 SMF3 102 99 27 25 14 33 58 (97%)
(57%)
[0235] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0236] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
* * * * *