U.S. patent application number 10/869324 was filed with the patent office on 2005-04-21 for glyphosate resistant maize lines.
Invention is credited to Gwyn, J. Jefferson, McElroy, David, Mumm, Rita, Spencer, Michael, Stephens, Michael A..
Application Number | 20050086719 10/869324 |
Document ID | / |
Family ID | 27420226 |
Filed Date | 2005-04-21 |
United States Patent
Application |
20050086719 |
Kind Code |
A1 |
Spencer, Michael ; et
al. |
April 21, 2005 |
Glyphosate resistant maize lines
Abstract
Methods and compositions relating to glyphosate resistant maize
plants, including the GA21, GG25, GJ11 and FI117 transformation
events, are disclosed. Also disclosed are methods of using
herbicide resistance transformation events in plant breeding
procedures. The invention further includes methods of ensuring
plant seed purity.
Inventors: |
Spencer, Michael; (Mystic,
CT) ; Mumm, Rita; (Tolono, IL) ; Gwyn, J.
Jefferson; (Mahomet, IL) ; McElroy, David; (N.
Stonington, CT) ; Stephens, Michael A.; (East Lyme,
CT) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI L.L.P.
600 CONGRESS AVE.
SUITE 2400
AUSTIN
TX
78701
US
|
Family ID: |
27420226 |
Appl. No.: |
10/869324 |
Filed: |
June 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10869324 |
Jun 16, 2004 |
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09698789 |
Oct 27, 2000 |
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6762344 |
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09698789 |
Oct 27, 2000 |
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08927368 |
Sep 11, 1997 |
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08927368 |
Sep 11, 1997 |
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08899247 |
Jul 23, 1997 |
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08899247 |
Jul 23, 1997 |
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08832078 |
Apr 3, 1997 |
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6040497 |
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Current U.S.
Class: |
800/300 |
Current CPC
Class: |
C12N 9/1092 20130101;
C12N 15/8275 20130101; C12N 15/8289 20130101; A01H 1/04
20130101 |
Class at
Publication: |
800/300 |
International
Class: |
A01H 005/00 |
Claims
1-108. (canceled)
109. A method of testing the quality of plant seeds comprising a
transformation event conferring resistance to a preselected
herbicide, the method comprising the steps: (i) planting said
seeds; (ii) cultivating said seeds; (iii) treating the plants grown
from said seeds with said preselected herbicide; and (iv)
identifying plants which are resistant to said herbicide.
110-118. (canceled)
Description
[0001] This application is a continuation-in-part of application
Ser. No. ______ filed Jul. 27, 1997; which is a
continuation-in-part of application Ser. No. 08/832,078 filed Apr.
3, 1997.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to transgenic maize
plants which are resistant to the herbicides and methods of using
same. More specifically, it relates to the maize transformation
events GA21, GG25, FI117 and GJ11.
[0004] 2. Description of the Related Art
[0005] Chemical weed control is a powerful tool of our
technological age. Long known as one of the most arduous of
agricultural operations, weed killing has taken on an entirely new
aspect as chemical after chemical is added to the arsenal of
herbicides. The U.S. has led the world both in production and use
of herbicides and as a result yields of maize, soybeans, cotton,
sugar beets, and many other crops have increased since 1945, in
some cases 100% or more. Thus while use of fertilizers and new
high-yielding crop varieties have contributed greatly to the "green
revolution" chemical weed control has been at the forefront in
technological achievement.
[0006] A particularly useful type of herbicide is one having a
broad spectrum of herbicidal activity. Use of such herbicides
obviates the need for application of multiple herbicides. The
problem with such herbicides is that they typically have a
deleterious effect on any crops which are exposed to the herbicide.
One way to overcome this is to produce transformed crop plants with
genes which confer resistance to certain broad spectrum
herbicides.
[0007] Recent advances in genetic engineering have provided the
requisite tools to transform plants to contain foreign genes.
Plants may, therefore, be produced which have unique
characteristics of agronomic importance. Certainly, weed control
via herbicide tolerance is one such advantageous trait which is
highly cost effective and environmentally compatible.
Herbicide-tolerant plants may reduce the need for tillage to
control weeds, thereby effectively reducing soil erosion. Further,
herbicide resistant plants can reduce the number of different
herbicides applied in the field.
[0008] One herbicide which is the subject of much investigation in
this regard is N-phosphonomethyl-glycine, commonly referred to as
glyphosate. Glyphosate inhibits the shikimic acid pathway which
leads to the biosynthesis of aromatic compounds including amino
acids and vitamins. Specifically, glyphosate inhibits the
conversion of phosphoenolpyruvic acid and 3-phosphoshikimic acid to
5-enolpyruvyl-3-phosphoshikimic acid by inhibiting the enzyme
5-enolpyruvyl-3-phosphoshikimic acid synthase (EPSP synthase or
EPSPS).
[0009] It has been shown that glyphosate tolerant plants can be
produced by introducing, into the genome of the plant, the capacity
to produce a higher level of EPSP synthase which enzyme is
preferably glyphosate tolerant (Shah et al., 1986). The
introduction into plants of glyphosate degradation gene(s) can
provide a means of conferring glyphosate tolerance to plants and/or
to augment the tolerance of transgenic plants already expressing a
glyphosate tolerant EPSP synthase depending upon the physiological
effects of the degradation products.
[0010] Glyphosate metabolism (degradation) has been examined in a
wide variety of plants and little degradation has been reported in
most of those studies. In those instances where degradation has
been reported, the initial breakdown product is usually
aminomethylphosphonate (AMPA) (Coupland, 1985; Marshall et al.,
1987). In these instances, it is not clear if glyphosate is
metabolized by the plant or by the contaminating microbes on the
leaf surface to which glyphosate was applied. AMPA has been
reported to be much less phytotoxic than glyphosate for most plant
species (Franz, 1985) but not for all plant species (Maier, 1983;
Tanaka et al., 1986). Glyphosate degradation in soils is much more
extensive and rapid (Torstensson, 1985). The principal breakdown
product identified is AMPA (Rueppel et al., 1977; Nomura and
Hilton. 1977); a phosphonate that can be metabolized by a wide
variety of microorganisms (Zeleznick et al., 1963; Mastalerz et
al., 1965; Cook et al., 1978; Daughton et al., 1979a; 1979b; 1979c;
Wackett et al., 1987a). A number of pure cultures of bacteria have
been identified that degrade glyphosate by one of the two known
routes (Schowanek and Verstraete, 1990; Weidhase et al., 1990; Liu
et al., 1991). A route involving a "C--P lyase" that degrades
glyphosate to sarcosine and inorganic orthophosphate (Pi) has been
reported for a Pseudomonas sp. (Shinabarger and Braymer, 1986;
Kishore and Jacob, 1987) and an Arthrobacter sp. (Pipke et al.,
1987b). Pure cultures capable of degrading glyphosate to AMPA have
been reported for a Flavobacterium sp. (Balthazor and Hallas,
1986), for a Pseudomonas sp. (Jacob et al., 1988) and for
Arthrobacter atrocyaneus (Pipke and Amrhein, 1988). In addition, a
large number of isolates that convert glyphosate to AMPA have been
identified from industrial activated sludges that treat glyphosate
wastes (Hallas et al., 1988). However, the number and nature of
bacterial genes responsible for these degradations have not been
heretofore determined nor have the gene(s) been isolated.
[0011] The development of plants resistant to the herbicidal
compound glyphosate has been a goal in the engineering of many
plant species (U.S. Pat. No. 4,769,061). The development of
glyphosate resistant tobacco plants was reported by Comai et al.,
(1985). Herbicide resistance was conferred on plants by expression
of an aroA gene derived from Salmonella typhimurium encoding a
glyphosate resistant form of the enzyme EPSP synthase. In addition,
glyphosate resistant soybeans were produced (Monsanto, APHIS
petition 93-258-01p). Methods for production of glyphosate
resistant corn plants also have been described (WO 95/06128; U.S.
Pat. No. 5,554,798). Similarly, a glyphosate oxidoreductase gene
has been described for use in conferring glyphosate resistance
(U.S. Pat. No. 5,463,175).
[0012] The ultimate goal in producing transgenic glyphosate
resistant maize plants is to provide plants which may be treated
with glyphosate at a level sufficient for killing weeds, without a
deleterious effect on yield or fertility. In this respect, the
prior art has failed. There is, therefore, a great need in
agriculture for maize plants which can be directly sprayed in the
field with glyphosate, thereby killing weeds, but otherwise not
producing a deleterious effect on the crop itself.
SUMMARY OF THE INVENTION
[0013] The present invention seeks to overcome deficiencies in the
prior art by providing fertile transgenic maize plants which can be
treated with glyphosate in the field without a resulting loss in
yield or fertility. Therefore, one aspect of the present invention
relates to a fertile transgenic maize plant comprising a
chromosomally incorporated expression cassette. In particular
embodiments the expression cassette comprises: (i) a modified maize
EPSPS gene encoding an EPSPS product having isoleucine at position
102 and serine at position 106, and (ii) a promoter active in maize
operably linked to said EPSPS gene, wherein the yield of said
fertile transgenic maize plant is not affected by a glyphosate
application rate that affects the yield of a maize plant lacking
said modified maize gene.
[0014] In another aspect, the maize plant may comprise a promoter
which is selected from the group consisting of a rice actin
promoter, a maize histone promoter and a fused CaMV
.sup.35S-Arabidopsis histone promoter. In one embodiment, the plant
may comprise an expression cassette which is derived from pDPG434,
pDPG427 or pDPG443. The expression cassette may, in particular
embodiments, be further be defined as pDPG434, and the maize plant
may be further defined as comprising a transformation event
selected from the group consisting of GA21 and FI117; seeds
comprising these events having been deposited with the ATCC and
assigned the ATCC accession numbers ATCC 209033, and ATCC 209031,
respectively. The maize plant comprising the FI117 transformation
event may further be defined as comprising a bar gene.
[0015] In yet another aspect, the maize plant may comprise a
pDPG427 expression cassette and may be further defined as
comprising the transformation event GG25 or, may comprise an
expression cassette of pDPG443 and the maize plant may be further
defined as comprising the transformation event GJ11; seeds
comprising the GG25 and GJ11 transformation events having been
deposited with the ATCC and assigned the ATCC accession numbers
ATCC 209032 and ATCC 209030, respectively. The invention is
intended to include the progeny of any generation and seeds of the
above maize plants, as well as the seeds of the progeny of any
generation.
[0016] Still yet another aspect of the current invention comprises
a method of preparing a fertile transgenic maize plant. The method
comprises: (i) providing an expression cassette comprising (a) a
modified maize EPSPS gene encoding an EPSPS product having
isoleucine at position 102 and serine at position 106 and (b) a
promoter active in maize operably linked to said EPSPS gene; (ii)
contacting recipient maize cells with said expression cassette
under conditions permitting the uptake of said expression cassette
by said recipient cells; (iii) selecting recipient cells comprising
a chromosomally incorporated expression cassette; (iv) regenerating
plants from said selected cells; and (v) identifying a fertile
transgenic maize plant, the yield of which is not affected by a
glyphosate application rate that affects the yield of a maize
lacking said modified maize gene.
[0017] The method may comprise any method of contacting including,
but not limited to, microprojectile bombardment, electroporation,
or Agrobacterium-mediated transformation. Said selecting may
comprise treating recipient cells with glyphosate. The promoter may
be selected from the group consisting of a rice actin promoter, a
maize histone promoter and a fused CaMV 35S-Arabidopsis histone
promoter. In particular embodiments, said expression cassette may
be derived from pDPG434, pDPG427 and/or pDPG443. The expression
cassette may, in particular, be pDPG434 and the maize plant may be
further defined as comprising a transformation event selected from
the group consisting of GA21 and FI117. In the method, the
transformation event may also be FI117, and said maize plant may
further defined as comprising a bar gene. The expression cassette
may also be pDPG427, and the maize plant may be further defined as
comprising the transformation event GG25. The method also includes
an expression cassette of pDPG443 where the maize plant may be
further defined as comprising the transformation event GJ11.
[0018] In still yet another aspect, the invention is a fertile
transgenic maize plant prepared according to a method comprising:
(i) providing an expression cassette comprising (a) a modified
maize EPSPS gene encoding an EPSPS product having isoleucine at
position 102 and serine at position 106 and (b) a promoter active
in maize operably linked to said EPSPS gene; (ii) contacting
recipient maize cells with said expression cassette under
conditions permitting the uptake of said expression cassette by
said recipient cells; (iii) selecting recipient cells comprising a
chromosomally incorporated expression cassette; (iv) regenerating
plants from said selected cells; and (v) identifying a fertile
transgenic maize, the yield of which is not affected by a
glyphosate application rate that affects the yield of a maize
lacking said modified maize gene. The maize may have a promoter
selected from the group consisting of a rice actin promoter, a
maize histone promoter and a fused CAMV .sup.35S-Arabidopsis
histone promoter. The expression cassette may be derived from
pDPG434, pDPG427 and pDPG443. The invention includes progeny of any
generation and seeds of the fertile transgenic maize plant, as well
as seeds of the progeny of the maize plant.
[0019] Still yet another aspect of the current invention is a
glyphosate resistant, inbred, fertile maize plant comprising a
chromosomally incorporated expression cassette comprising (a) a
modified maize EPSPS gene encoding an EPSPS product having
isoleucine at position 102 and serine at position 106 and (b) a
promoter active in maize operably linked to said EPSPS gene. The
promoter may be selected from the group consisting of a rice actin
promoter, a maize histone promoter and a fused CaMV
.sup.35S-Arabidopsis histone promoter. The expression cassette may
be derived from pDPG434, pDPG427 and pDPG443. In particular
embodiments the inbred maize plant may be further defined as
comprising a transformation event selected from the group
consisting of GJ11, FI117, GG25 or GA21, seeds comprising these
transformation events having been deposited and assigned the ATCC
accession numbers ATCC 209030, ATCC 209031, ATCC 209032, and ATCC
209033, respectively.
[0020] Still yet another aspect of the current invention is a
glyphosate resistant, crossed fertile transgenic maize plant
prepared according to the method comprising: (i) obtaining a
fertile transgenic maize plant comprising a chromosomally
incorporated expression cassette comprising (a) a modified maize
EPSPS gene encoding an EPSPS product having isoleucine at position
102 and serine at position 106 and (b) a promoter active in maize
operably linked to said EPSPS gene; (ii) crossing said fertile
transgenic maize plant with a second maize plant lacking said
expression cassette to obtain a third maize plant comprising said
expression cassette; and (iii) backcrossing said third maize plant
to obtain a backcrossed fertile maize plant; wherein said modified
EPSPS gene is inherited through a male parent. In particular
embodiments the second maize plant is an inbred. The third maize
plant may be a hybrid. The maize plant may, in particular
embodiments be further defined as comprising a transformation event
selected from the group consisting of GJ11, FI117, GG25 or GA21,
ATCC accession numbers ATCC 209030, ATCC 209031, ATCC 209032, and
ATCC 209033, respectively.
[0021] Still yet another embodiment of the invention is a
glyphosate resistant, crossed fertile transgenic maize plant
prepared according to the method comprising: (i) obtaining a
fertile transgenic maize plant comprising a chromosomally
incorporated expression cassette comprising (a) a modified maize
EPSPS gene encoding an EPSPS product having isoleucine at position
102 and serine at position 106 and (b) a promoter active in maize
operably linked to said EPSPS gene; and (ii) crossing said fertile
transgenic maize plant with a second maize plant lacking said
expression cassette to obtain a third maize plant comprising said
expression cassette; wherein said modified EPSPS gene is inherited
through a female parent. In particular embodiments, the second
maize plant may be an inbred, and the third maize plant may be a
hybrid. The maize plant may, in particular embodiments, be further
defined as comprising a transformation event selected from the
group consisting of GJ11, FI117, GG25 or GA21, seeds comprising
these transformation events having been deposited and assigned the
ATCC accession numbers ATCC 209030, ATCC 209031, ATCC 209032, and
ATCC 209033, respectively.
[0022] Still yet another aspect of the invention is a glyphosate
resistant, crossed fertile transgenic maize plant prepared
according to the method comprising: (i) obtaining a fertile
transgenic maize plant comprising a chromosomally incorporated
expression cassette comprising (a) a modified maize EPSPS gene
encoding an EPSPS product having isoleucine at position 102 and
serine at position 106 and (b) a promoter active in maize operably
linked to said EPSPS gene; (ii) crossing said fertile transgenic
maize plant with a second maize plant to obtain a third maize plant
comprising said expression cassette; and (iii) backcrossing said
third maize plant to obtain a backcrossed fertile maize plant;
wherein said modified EPSPS gene is inherited through a female
parent. In particular embodiments, the maize plant may be an inbred
and the third maize plant may be a hybrid. In one embodiment the
maize plant may be further defined as comprising a transformation
event selected from the group consisting of a GJ11, FI117, GG25 or
GA21 transformation event, seeds comprising these transformation
events having the ATCC accession numbers ATCC 209030, ATCC 209031,
ATCC 209032, and ATCC 209033, respectively.
[0023] Still yet another aspect of the current invention is a
glyphosate resistant, hybrid maize plant comprising a chromosomally
incorporated expression cassette comprising (a) a modified maize
EPSPS gene encoding an EPSPS product having isoleucine at position
102 and serine at position 106 and (b) a promoter active in maize
operably linked to said EPSPS gene. In one embodiment, the promoter
is selected from the group consisting of a rice actin promoter, a
maize histone promoter and a fused CaMV 35S-Arabidopsis histone
promoter and the expression cassette is derived from pDPG434,
pDPG427 and pDPG443. The maize plant may, in particular
embodiments, be further defined as comprising a transformation
event selected from the group consisting of GA21, GG25, GJ11 and
FI117.
[0024] Still yet another aspect of the invention is a glyphosate
resistant, hybrid, transgenic maize plant prepared according to the
method comprising crossing a first and second inbred maize plant,
wherein one of said first and second inbred maize plants comprises
a chromosomally incorporated expression cassette comprising (a) a
modified maize EPSPS gene encoding an EPSPS product having
isoleucine at position 102 and serine at position 106 and (b) a
promoter active in maize operably linked to said EPSPS gene. In one
embodiment, the promoter is selected from the group consisting of a
rice actin promoter, a maize histone promoter and a fused CaMV
35S-Arabidopsis histone promoter, and said expression cassette is
derived from pDPG434, pDPG427 and/or pDPG443. The maize plant may,
in particular embodiments, be further defined as comprising a
transformation event selected from the group consisting of GA21,
GG25, GJ11 and FI117.
[0025] Still yet another aspect of the invention is a glyphosate
resistant, crossed fertile transgenic maize plant prepared by a
process comprising: (i) obtaining a fertile transgenic maize plant
comprising a chromosomally integrated expression cassette
comprising (a) a modified maize EPSPS gene encoding an EPSPS
product having isoleucine at position 102 and serine at position
106 and (b) a promoter active in maize operably linked to said
EPSPS gene; (ii) crossing said fertile transgenic maize plant with
a second maize plant to obtain a third maize plant comprising said
expression cassette; and (iii) crossing said third fertile
transgenic maize plant with a fourth maize plant to obtain a fifth
transgenic maize plant comprising said expression cassette. In one
embodiment, the second and fourth maize plants have the same
genotype. In another embodiment the second and fourth maize plants
have different genotypes.
[0026] Still yet another aspect of the invention is seed of a
fertile, transgenic maize plant, said seed comprising a
chromosomally incorporated expression cassette comprising (a) a
modified maize EPSPS gene encoding an EPSPS product having
isoleucine at position 102 and serine at position 106 and (b) a
promoter active in maize operably linked to said EPSPS gene, said
seed prepared by a process comprising the steps of: (i) obtaining a
parental fertile, transgenic maize plant comprising a chromosomally
incorporated expression cassette comprising (a) a modified maize
EPSPS gene encoding an EPSPS product having isoleucine at position
102 and serine at position 106 and (b) a promoter active in maize
operably linked to said EPSPS gene; (ii) breeding said parental
plant with a second fertile maize plant to produce a plurality of
progeny fertile, transgenic maize plants, said progeny maize plants
including plants that express a chromosomally incorporated
expression cassette comprising (a) a modified maize EPSPS gene
encoding an EPSPS product having isoleucine at position 102 and
serine at position 106 and (b) a promoter active in maize operably
linked to said EPSPS gene; (iii) selecting from said progeny maize
plants a plant having resistance to glyphosate; and (iv) obtaining
seed from said selected progeny maize plant. In one embodiment the
progeny maize plants are two generations removed from the parental
transgenic maize plant.
[0027] The progeny maize plants having resistance to glyphosate may
be selected by testing plants for resistance to glyphosate at an
application rate of, for example 1.times., 2.times., 3.times. or
4.times. (1.times. is equivalent to 16 ounces of Roundup.TM. per
acre). In a particular embodiment, the second fertile maize plant
is a non-transgenic maize plant and the plant is pollinated with
pollen from a male parental transgenic maize plant. The parental
maize plant may be pollinated with pollen from said second fertile
maize plant and wherein said parental maize plant is a female
parental transgenic maize plant.
[0028] Still yet another aspect of the invention is a method of
increasing the yield of corn in a field comprising: (i) planting
fertile transgenic maize plants transformed with an expression
cassette comprising (a) a modified maize EPSPS gene encoding an
EPSPS protein having isoleucine at position 102 and serine at
position 106 and (b) a promoter active in maize operably linked to
said EPSPS gene; and (ii) applying glyphosate to said field at an
application rate that inhibits the yield of a maize plant that does
not comprise said modified maize gene, wherein the yield of said
fertile transgenic maize plant is not affected by said glyphosate
application. In particular embodiments, the glyphosate application
rate may be 1.times., 2.times. or 4.times..
[0029] Still yet another aspect of the invention is a method of
inhibiting weed growth in a corn field comprising: (i) planting
fertile transgenic maize plants transformed with an expression
cassette comprising (a) a modified maize EPSPS gene encoding an
EPSPS protein having isoleucine at position 102 and serine at
position 106 and (b) a promoter active in maize operably linked to
said EPSPS gene; and (ii) applying glyphosate to said field at an
application rate that inhibits the yield of a maize plant that does
not comprise said modified maize gene, wherein the yield of said
fertile transgenic maize plant is not affected by said glyphosate
application. In particular embodiments, the glyphosate application
rate may be 1.times., 2.times., or 4.times..
[0030] Still yet another aspect of the invention is a method of
growing corn comprising: (i) planting fertile transgenic maize
plants transformed with an expression cassette comprising (a) a
modified maize EPSPS gene encoding an EPSPS protein having
isoleucine at position 102 and serine at position 106 and (b) a
promoter active in maize operably linked to said EPSPS gene; and
(ii) treating said corn with glyphosate at an application rate that
inhibits the yield of a maize plant that does not comprise said
modified maize gene, wherein the yield of said fertile transgenic
maize plant is not affected by said glyphosate application. In
particular embodiments, the application rate may be, 1.times.,
2.times. or 4.times..
[0031] It is clear that the ability to provide even a single
fertile, transgenic corn line is generally sufficient to allow the
introduction of the transgenic component (e.g., recombinant DNA) of
that line into a second corn line of choice. This is because by
providing fertile, transgenic offspring, the practice of the
invention allows one to subsequently, through a series of breeding
manipulations, move a selected gene from one corn line into an
entirely different corn line. Therefore, the current invention is
intended to include any maize plant, from any generation, which has
one or more transgenes comprising a GJ11, FI117, GG25 or GA21
transformation event; seeds comprising these transformation events
having the ATCC accession numbers ATCC 209030, ATCC 209031, ATCC
209032, and ATCC 209033, respectively. The invention further
includes the seeds of maize plants of any generation comprising the
GJ11, FI117, GG25 or GA21 transformation events.
[0032] Still yet aspect of the invention is a method for producing
animal feed. This method may include the steps of (i) obtaining a
fertile transgenic maize plant comprising a chromosomally
integrated expression cassette comprising (a) a modified maize
EPSPS gene encoding an EPSPS protein having isoleucine at position
102 and serine at position 106 and (b) a promoter active in maize
operably linked to the EPSPS gene; (ii) cultivating the transgenic
Zea mays plant; (iii) obtaining seed from the cultivated Zea mays
plant; and (iv) preparing animal feed from said seed. In particular
embodiments, the fertile transgenic maize plants are further
defined as comprising DNA from a plasmid selected from the group
consisting of pDPG434, pDPG427 and pDPG443. In further embodiments,
the fertile transgenic maize plants will comprise a transformation
event selected from the group consisting of: GJ11, GG25, FI117 and
GA21.
[0033] Still yet another aspect of the current invention is a
method for producing food comprising the steps of: (i) obtaining a
fertile transgenic Zea mays plant comprising heterologous DNA
comprising a transformation event selected from the group
consisting of GG25, GJ11, FI117 and GA21, wherein the DNA is
heritable; (ii) cultivating the transgenic Zea mays plant; (iii)
obtaining seed from the cultivated Zea mays plant; and (iv)
preparing human food from the seed. Also included in the current
invention is a method for producing oil comprising: (i) obtaining a
fertile transgenic Zea mays plant comprising heterologous DNA
comprising a transformation event selected from the group
consisting of GG25, GJ11, FI117 and GA21, wherein the DNA is
heritable; (ii) cultivating the transgenic Zea mays plant; (iii)
obtaining seed from the cultivated Zea mays plant; and (iv)
preparing oil from the seed.
[0034] Still yet another aspect of the current invention is a
method for producing starch comprising the steps: (i) obtaining a
fertile transgenic Zea mays plant comprising heterologous DNA
comprising a transformation event selected from the group
consisting of GG25, GJ11, FI117 and GA21, wherein the DNA is
heritable; (ii) cultivating said transgenic Zea mays plant; (iii)
obtaining seed from the cultivated Zea mays plant; and (iv)
preparing starch from the seed.
[0035] Still yet another aspect of the current invention is a
method for producing seed comprising: (i) obtaining a fertile
transgenic maize plant comprising a chromosomally integrated
expression cassette comprising (a) a modified maize EPSPS gene
encoding an EPSPS protein having isoleucine at position 102 and
serine at position 106 and (b) a promoter active in maize operably
linked to said EPSPS gene; (ii) cultivating said transgenic Zea
mays plant; and (iii) obtaining seed from said cultivated Zea mays
plant.
[0036] Still yet another aspect of the current invention provides a
method of plant breeding comprising the steps of: (i) planting in
pollinating proximity seeds capable of growing into first and
second parent plants, wherein the first parent plant comprises a
first transgene, the plant being able to be rendered male-sterile
by treatment with a preselected herbicide, and wherein the first
plant is resistant to said preselected herbicide; (ii) cultivating
the seeds to produce the first and second parent plants; (iii)
inducing male-sterility in the first parent plant by treating the
plant with the preselected herbicide; (iv) allowing the second corn
plant to pollinate the first parent plant; and (v) collecting seeds
produced on the first plant. In particular embodiments the second
parent plant is further defined as being resistant to the
preselected herbicide.
[0037] The first and second plants may be selected from the group
consisting of maize, wheat, rice, oat, barley, sorghum, sunflower,
alfalfa and soybean. The preselected herbicide may be glyphosate,
however, in other embodiments the herbicide may be glufosinate,
imidazolinone, sulphonylurea, kanamycin, G418, bromoxynil or
methotrexate. The first transgene may comprise a GG25
transformation event and/or a GJ11 transformation event, or any
other suitable, similar transgene. The second plant may comprise a
GA21 transformation event and/or a FI117 transformation event, or
any other suitable, similar transgene. In particular embodiments
the step of inducing male-sterility comprises applying a
concentration of glyphosate of from 8 ounces per acre to 96 ounces
per acre, which may be applied between the V5 and VT stages of
development.
[0038] Still yet another aspect of the current invention is a
method of testing seed quality of a hybrid maize seed comprising a
herbicide resistance transformation event, such as GA21, GG25,
FI117 or GJ11. The method comprises the steps of: (i) planting said
seed; (ii) cultivating the seed; and (iii) treating the plants
grown from the seed with a preselected herbicide. In particular
embodiments the seeds are selected from the group consisting of
maize seeds, wheat seeds, rice seeds, oat seeds, barley seeds,
sorghum seeds, sunflower seeds, alfalfa seeds and soybean seeds. In
other embodiments the seeds are maize seeds. The transformation
event may comprise a mutated EPSPS and the preselected herbicide
may be glyphosate. More specifically, the plants may be treated
with from 8 to 96 ounces per acre of glyphosate, and this treatment
may take place between the V4 and VT stages of development.
Alternatively the gene may be another suitable herbicide resistance
gene and the preselected herbicide selected from the group
consisting of glufosinate, imidazolinone, sulphonylurea, kanamycin,
G418, bromoxynil and methotrexate.
[0039] Still yet another aspect of the invention is a method of
plant breeding comprising the steps: (i) planting a seed capable of
growing into a first plant, the plant comprising a transformation
event conferring herbicide resistance; (ii) cultivating the seed to
produce the first plant; (iii) treating the first plant with a
preselected herbicide to render pollen not having the
transformation event inviable; (iv) allowing pollen having the
transformation event to pollinate the first plant or a second
plant, wherein the pollen having the transformation event remains
viable following the treating; and (v) collecting seed from the
first or the second plant. The transformation event may comprise a
mutated EPSPS gene operably linked to a promoter functional in said
first plant, and may further be a GA21 or FI117. Treating the first
maize plant may comprise treating the first maize plant with from 8
to 96 ounces per acre of glyphosate, and may take place between the
V4 and VT stages of development. The first plant may be selected
from the group consisting of maize, wheat, rice, oat, barley,
sorghum sunflower, alfalfa, and soybean. In addition to glyphosate,
the preselected herbicide may also be selected from the group
consisting of glufosinate, imidazolinone, sulphonylurea, kanamycin,
G418, bromoxynil and methotrexate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1. Plasmid map of pDPG165. Restriction sites are shown
and locations are indicated in base pairs.
[0041] FIG. 2. Plasmid map of pDPG427. Restriction sites used for
Southern blot analyses are shown and locations are indicated in
base pairs.
[0042] FIG. 3. Plasmid map of pDPG434. Restriction sites used for
Southern blot analyses are shown and locations are indicated in
base pairs.
[0043] FIG. 4. Plasmid map of pDPG443. Restriction sites used for
Southern blot analyses are shown and locations are indicated in
base pairs.
[0044] FIGS. 5A and 5B. Southern blot analysis to determine the
number of transgene insertions in GA21. A: Lane 1 contains GA21 DNA
digested with EcoRV. Lane 2 contains non-transformed control DNA
digested with EcoRV. Lane 3 contains pDPG434 digested with NotI.
The blot was probed with the 3.4 kb NotI fragment from pDPG434. B:
The blot shown in A was stripped and reprobed with a 324 bp
fragment of the mutant EPSPS gene.
[0045] FIG. 6. Southern blot analysis to estimate the copy number
and integrity of the mutant EPSPS Gene. Lane 1 contains GA21 DNA
digested with EcoRI/XbaI. Lane 2 contains nontransformed control
DNA digested with EcoRI/XbaI. Lane 3 contains pDPG434 digested with
EcoRI/XbaI. The blot was probed with the 324 bp EPSPS gene PCR
fragment.
[0046] FIG. 7. Southern blot analysis to confirm the lack of
plasmid backbone sequence in GA21. Genomic DNA of a bla gene
transformed plant (lane 1), a GA21 plant (lane 2), and plasmid DNA
of pDPG427 was digested with BglII The blot was probed with a 1.7
SspI/AflIII kb fragment from pBluescript SK(-) that contains the
ColE1 origin of replication and the bla gene.
[0047] FIGS. 8A and 8B. Effect of glyphosate application on the
growth and fertility of DK580 and DK626 BC.sub.4 hebrids of GA21.
FI117. GG25 and GJ11 transformation events. Treatments consisted of
glyphosate applications at the 0.times., 1.times. and 4.times.
rates (1.times.=16 ounces of ROUNDUP ULTRA.TM./acre). Mean ELH
(extended leaf height in centimeters) was measured 10 days after
glyphosate application. A. Effects of glyphosate application at the
V4 stage of development. B. Effects of glyphosate application at
the V8 stage of development.
[0048] FIGS. 9A and 9B. Yield effect of glyphosate application on
DK580 and DK626 hybrids with the FI117, GA21. GG25 and GJ11
transformation events. Comparisons are made between the 4
transformation events in each of the two hybrids both with and
without glyphosate application. Additionally, comparisons are made
between each of the hybrids with the introgressed transformation
event versus the hybrid without the transformation event. A.
Comparisons of effect of glyphosate application on the yield of
DK580 hybrids when applied at V4. B. Effect of glyphosate
application on the yield of DK626 hybrids when applied at V8.
[0049] FIG. 10. Southern blot analysis to detect transgene
insertions GA21. FI117. GG25 and GJ11. Southern blot of BglII
digested genomic DNA (lanes 2, 5, 10, 11, 12) and plasmid DNA (lane
13). Blot was probed with the 0.27 kb nos 3' polyadenlylation
region from the nopaline synthase gene of Agrobacterium tumefaciens
(Bevan, 1984). Lanes 2, 5, 10 and 11 contain genomic DNA from
plants having the FI117, GA21, GG25 and GJ11 transformation events,
respectively. Lane 12 contains negative control DNA from a
non-transformed maize plant and lane 13 contains pDPG427 plasmid
DNA.
[0050] FIGS. 11A, 11B, and 11C. Southern blot analysis to detect
transgene insertions GA21. GG25 and GJ11 using various restriction
enzymes. Genomic DNA of a non-transformed control plant (lane 1) as
well as GA21, GG25 and GJ11 (lanes 2, 3 and 4, respectively)
transformation event containing plants was digested with various
restriction enzymes and probed with a PCR generated 324 bp fragment
of the EPSPS gene (see example 8 for generation of EPSPS fragment).
DNA was digested with EcoRI (FIG. 11A), SphI (FIG. 11B) and SacI.
(FIG. 11C).
[0051] FIG. 12. Deduced amino acid sequence of the mutant corn
EPSPS Protein. Sequence includes the OTP transit peptide isolated
from corn and sunflower ribulose-1,5-bis phosphate carboxylase
oxygenase (RuBis Co) genes, (amino acids 1-125 are the transit
peptide).
[0052] FIG. 13. Field layout for study of glyphosate resistance in
GA21, GG25, FI117 and GJ11 DK580 and DK626 hybrids. The repetition
(1-3), column (COL1-COL12), row (14), hybrid (DK580 or DK626),
transformation event (GA21, FI117, GG25, or GJ11), transformed or
non-transformed status (N or T), glyphosate application level
(0.times., 1.times. or 4.times.), and developmental stage at
glyphosate application (V4 or V8), are given. Tests were conducted
in Dekalb, Ill., and Thomasboro, Ill. during 1996. All rows were
planted at double normal planting density, i.e., 60 seeds per row,
because hybrids segregated 1:1 for the glyphosate resistance trait.
Sprayed plants were thinned to 30 plants per row no sooner than 7
days after application of glyphosate at a time when glyphosate
susceptible plants could be identified. Unsprayed plots were
thinned to 30 plants per row at the same time.
[0053] FIG. 14. Plasmid map of pDPG425. Major components and
restriction sites are shown and locations are indicated in kilobase
pairs.
[0054] FIG. 15. Plasmid map of pDPG405. Major components and
restriction sites are shown and locations are indicated in base
pairs.
DETAILED DESCRIPTION OF THE INVENTION
[0055] In addition to direct transformation of a particular
genotype with a mutant EPSPS gene, glyphosate resistant plants may
be made by crossing a plant having a mutant EPSPS gene to a second,
glyphosate sensitive plant. "Crossing" a plant to provide a plant
line having an increased yield relative to a starting plant line,
as disclosed herein, is defined as the techniques that result in a
mutant EPSPS gene being introduced into a plant line by crossing a
starting line with a donor plant line that comprises a mutant EPSPS
gene. To achieve this one would, generally, perform the following
steps:
[0056] (a) plant seeds of the first (starting line) and second
(donor plant line that comprises a mutant EPSPS gene) parent
plants;
[0057] (b) grow the seeds of the first and second parent plants
into plants that bear flowers;
[0058] (c) pollinate the female flower of the first parent plant
with the pollen of the second parent plant; and
[0059] (d) harvest seeds produced on the parent plant bearing the
female flower.
[0060] Backcross conversion is herein defined as the process
including the steps of:
[0061] (a) crossing a plant of a first genotype containing a
desired gene, DNA sequence or element to a plant of a second
genotype lacking said desired gene, DNA sequence or element;
[0062] (b) selecting one or more progeny plant containing the
desired gene, DNA sequence or element;
[0063] (c) crossing the progeny plant to a plant of the second
genotype; and
[0064] (d) repeating steps (b) and (c) for the purpose of
transferring said desired gene, DNA sequence or element from a
plant of a first genotype to a plant of a second genotype.
[0065] Introgression of a DNA element into a plant genotype is
defined as the result of the process of backcross conversion. A
plant genotype into which a DNA sequence has been introgressed may
be referred to as a backcross converted genotype, line, inbred, or
hybrid. Similarly a plant genotype lacking said desired DNA
sequence may be referred to as an unconverted genotype, line,
inbred, or hybrid.
[0066] It is contemplated that glyphosate resistant plants may be
obtained by transfer of the DNA sequence comprising a mutant EPSPS
gene and adjacent plant genomic DNA sequences from FI117, GA21,
GG25 and GJ11 mutant EPSPS gene transformed donor plants to a
recipient plant whereby the recipient plant has increased tolerance
to the herbicide glyphosate following introduction of the mutant
EPSPS gene-encoding DNA segment. The DNA sequence may further be
transferred to other genotypes through the process of backcross
conversion and the glyphosate resistance of said backcross
converted plants, or hybrids derived therefrom, is increased
relative to the unconverted plant. The mutant EPSPS gene
integration events, as well as the associated vector DNA, may be
used as genetic markers in marker assisted breeding for the purpose
of selecting maize plants with increased herbicide resistance.
[0067] I. Herbicide Control of Weeds
[0068] Chemical weed control is a science that involves knowledge
in the fields of chemistry and biology, some familiarity with
reactions of plants to phytotoxic agents, and at least
observational experience in the responses of common weeds and crops
to herbicides. Weed and crop ecology and appreciation of the
factors determining selectivity, tolerance, and susceptibility are
important. And finally, one needs a vast backlog of detailed
information concerning the role of weed control in practical
agriculture.
[0069] Weeds pose a threat to human health and welfare. They reduce
the yield and value of crops; as well as increasing production and
harvesting costs. The principal means by which weeds cause these
effects are:
[0070] 1. Competing with crop plants for the essentials of growth
and development.
[0071] 2. Production of toxic or irritant chemicals that cause
human or animal health problems.
[0072] 3. Production of immense quantities of seed or vegetative
reproductive parts or both that contaminate agricultural products
and perpetuate the species in agricultural lands.
[0073] 4. Production on agricultural and nonagricultural lands of
vast amounts of vegetation that must be disposed of.
[0074] In nonagricultural areas, weeds are often considered more of
a nuisance than a threat; but even in this case weeds are a
potential human hazard. Weed pollen may cause hay fever or other
allergies, and toxic chemicals present in their sap or on their
leaves may cause skin irritations or rashes when brushed against.
Some substances produced by weeds are deadly when ingested. Weeds
tend to hide tools and equipment, switches and valves, irrigation
gates, and even holes in the ground. Dense, moisture-holding weed
growth aids in the deterioration of wooden structures and the
rusting of metal fences, buildings, and immobile machinery. Dead,
dry weeds constitute a fire hazard, subject to ignition by a spark,
a carelessly tossed cigarette, or even a piece of glass reflecting
sunlight. Weeds reduce the enjoyment of recreation areas. They
impede the flow of water in waterways and hamper water traffic
especially in tropical and subtropical regions.
[0075] In agricultural lands, weeds reduce crop yields and quality,
interfere with harvesting, and increase the time and costs involved
in crop production. Weeds harbor insects and plant disease
organisms; and in some cases, they serve as essential alternate
hosts for these pests. Some weeds are undesirable in hay, pastures,
and rangelands because of the mechanical injury that they inflict
on livestock. Woody stems, thorns, and stiff seed awns cause injury
to the mouth and digestive tract of livestock; and the hairs and
fibers of some plants tend to ball up and obstruct the intestines,
especially in horses, causing serious problems. Ingested by milk
cows, some weeds such as ragweeds, wild garlic (Allium vineale L.),
and mustard, among others, impart a distinctly distasteful odor or
flavor to milk and butter. Barbed seed dispersal units may become
so entangled in the wool of sheep as to greatly diminish its market
value. Parasitic plants, such as dodder (Cuscuta sp.), broomrape
(Orobanche sp.), and witchweed, rob their host plants of organic
foodstuffs.
[0076] Weeds may additionally serve as host plants for pests of
agriculture. Examples of weeds that serve as hosts for plant pests
are cited below. Pepperweed and tansymustard (Descurainia sp.)
maintain large populations of diamondback moths during the late
fall, winter, and spring; they are also hosts to the turnip aphid
and green peach aphid. Several weed species by the nightshade
family (Solanaceae) are hosts to insects that commonly attack
eggplant, pepper, potato, and tomato; for example, horsenettle
(Solanum carolinense L.) is a host of the Colorado potato beetle,
and black nightshade (S. nigrum L.) is a host of the cabbage
looper. Morning-glory is an important host of insects attacking
sweet potato, especially the highly destructive sweet potato
weevil. Ragweed serves as a host for Mansonia mosquitoes, an insect
vector for the human diseases encephalitis and rural filariasis.
European barberry (Berberis vulgaris L.) is an essential host of
the wheat stem rust in the northern wheat regions of the United
States. Goosegrass (Eleusine induce [L.] Great.) and purple
nutsedge are hosts of barley yellow dwarf virus. Currants and
gooseberries (Ribes sp.) are hosts for white pine blister rust.
[0077] One crop which is highly reliant on chemical control of
weeds is corn. Corn has been grown on 60 million to 83 million
acres per year in the period from 1982 to 1993. In 1993, fifteen
states had corn acreage in excess of one million acres, and 74% of
the crop was grown in Iowa, Illinois, Nebraska, Minnesota, and
Indiana. Herbicides were applied to about 97% of the corn acreage
in the United States, and over 98% of the corn acreage in Iowa,
Illinois, Minnesota, and Indiana had herbicide applications
(Agricultural Chemical Usage, 1994). Furthermore, an average of 2.1
active ingredients were applied per acre in 1992.
[0078] Weeds compete with corn for nutrients, water, and light and
when not controlled can significantly reduce the yield of corn. For
examples, it is estimated that between 1972 and 1976 corn yields
were reduced by about 10% due to weeds (Chandler, J. M., 1981, CRC
Handbook of Pest Management in Agriculture, Vol. I, edited by
Pimentel, D., pp. 95-109). It is especially important to control
weed growth early in corn plant development, because even small
numbers of weeds can have a dramatic negative impact on crop yield.
Weeds are primarily controlled by mechanical or chemical means.
Although mechanical cultivation is widely practiced, chemical weed
control measures are wide spread and greater than 95% of the corn
crop in the United States is treated with chemical herbicides.
Indiscriminate use of herbicides, however, can lead to development
of resistant weeds. Therefore it is important to develop methods of
chemical weed control that represent novel modes of action and are
unlikely to select for resistant weeds.
[0079] A diverse group of weed species necessitates a range of weed
control methods in corn. Broad leaf weeds such as velvetleaf,
pigweed, wild sunflowers, ragweed, and smartweed are of concern in
corn. Furthermore, grass weeds such as johnson grass, shattercane,
fall panicum, foxtails, quackgrass, wild proso millet and wooly
cupgrass are common in corn. Perennial weeds are an additional
problem as they are able to propagate by seed and/or underground
plants parts, and may necessitate multiple herbicide applications.
The wide array of weed species that are found in corn field
requires the use of multiple type of herbicides and multiple
applications in order to achieve weed control. Therefore, herbicide
application regimes vary depending on the weed spectrum and local
agronomic practices. Table 1 summarizes herbicide treatment of corn
acreage in 1993.
1TABLE 1 Herbicide Applications to Corn Percent of Acres Treated
with Major Corn Herbicides Major Corn Growing Herbicide Name States
(Including Minn.) Minnesota Atrazine 69 37 Metolachlor 32 24
Alachlor 24 23 Dicamba 21 48 Cyanazine 20 16 2,4-D 12 13 Bromoxynil
8 14 Nicosulfuron 6 19 Source: Agricultural Chemical Usage, March
1994, NASS and ERS, USDA.
[0080] A single application of herbicides near the time of planting
is most common for corn. Usually this application comprises one of
the triazine herbicides (atrazine, cyanazine, simazine) to control
broadleaf weeds and an acetanilide herbicide (metolachlor,
alachlor) to control annual grasses. Control of broadleaf weeds and
problem grasses with postemergent herbicides such as dicamba,
bromoxynil, bentazon, nicosulfuron and primisulfuron, occurred on
about half of the corn acreage in 1993. Choice of herbicide is
consistent in all but the north central states (e.g., Minnesota and
South Dakota). Atrazine was used on about 69% of the corn acreage
in 1993.
[0081] The most common tank mix was atrazine and metolachlor for
broad spectrum weed control. Herbicide usage in the north central
states, however, differs in that there is reduced usage of atrazine
due to carryover to small grains and soybeans in the high pH, low
rainfall soils of the region. Furthermore, because the growing
season is shorter in the north central region, postemergent
herbicides are preferred in that they do not delay planting
operations. For example, in 1993, the most common herbicide used on
corn in Minnesota was the postemergent herbicide dicamba (all data
from Agricultural Chemical Usage, 1994).
[0082] In selecting a herbicide for control of weeds in corn, a
chemical must be chosen that has a suitable spectrum of weeds that
are killed and will not have adverse long lasting effects on the
environment. In addition with increasing no till and minimum till
acreage for corn, it is necessary to have weed control agents
available that can be applied post-emergence and spot applied as
needed. Some of the herbicides currently applied to corn are
limited in weed spectrum, may persist in soil or contaminate ground
water, or may lead to the development of herbicide resistant weeds.
Moreover, some herbicides that have reduced potential for adverse
environmental effects and exhibit a broad spectrum of weed killing
ability are non-discriminatory in their plant killing ability, i.e.
crop plants such as corn are equally affected as weed species. It
is only through introduction of genes conferring resistance to such
herbicides that these chemicals can be used for weed control in
corn.
[0083] Glyphosate is a broad spectrum post-emergence herbicide that
is rapidly degraded in soil, has a low toxicity to non-target
organisms, and does not contribute to ground water contamination.
The availability of glyphosate for weed control in field grown corn
has previously been lacking because of the broad spectrum of its
effects. The glyphosate resistant transgenic plants described
herein will give the farmer increased flexibility in dealing with
weed problems. Glyphosate resistant corn hybrids will offer the
farmer 1) the use of a new herbicide which offers broad spectrum
control of annual and perennial, broad leaf and grass weeds; 2)
less dependence on pre-plant herbicide applications; 3) increased
flexibility in applying herbicides on an as needed basis; 4) a new
herbicidal mode of action which will decrease the likelihood of
development of herbicide resistant weeds; and 5) a herbicide for
use in no-till systems which conserve fuel and reduce soil erosion.
Because of the advantages offered, post-emergent herbicides are
being applied to increasing acreage of corn every year, e.g., about
15 million acres of corn, 20% of the total corn acreage, receive
only post-emergent herbicide applications. Glyphosate resistant
corn will provide the farmer with an alternative weed control
method. Currently on the average 2.1 herbicides are applied to corn
during the growing season. It is expected that the use of
glyphosate for weed control will reduce the number of kinds of
herbicides applied as well as the number of required applications.
Glyphosate resistant corn will, therefore, decrease the
environmental risks posed by herbicides while at the same time
increasing the efficacy of chemical weed control.
[0084] II. DNA Delivery
[0085] Following the generation of recipient cells, the present
invention generally next includes steps directed to introducing an
exogenous DNA segment into a recipient cell to create a transformed
cell. The frequency of occurrence of cells receiving DNA is
believed to be low. Moreover, it is most likely that not all
recipient cells receiving DNA segments will result in a transformed
cell wherein the DNA is stably integrated into the plant genome
and/or expressed. Some may show only initial and transient gene
expression. However, certain cells from virtually any monocot
species may be stably transformed, and these cells developed into
transgenic plants, through the application of the techniques
disclosed herein.
[0086] There are many methods for introducing transforming DNA
segments into cells, but not all are suitable for delivering DNA to
plant cells. Suitable methods are believed to include virtually any
method by which DNA can be introduced into a cell, such as by
Agrobacterium infection, direct delivery of DNA such as, for
example, by PEG-mediated transformation of protoplasts (Omirulleh
et al., 1993), by desiccation/inhibition-mediated DNA uptake, by
electroporation, by agitation with silicon carbide fibers, by
acceleration of DNA coated particles, etc. Agrobacterium-mediated
transformation of maize was described in U.S. Pat. No. 5,591,616,
which is specifically incorporated herein by reference. In certain
embodiments, acceleration methods are preferred and include, for
example, microprojectile bombardment and the like.
[0087] (i) Electroporation
[0088] Where one wishes to introduce DNA by means of
electroporation, it is contemplated that the method of Krzyzek et
al. (U.S. Pat. No. 5,384,253, incorporated herein by reference)
will be particularly advantageous. In this method, certain cell
wall-degrading enzymes, such as pectin-degrading enzymes, are
employed to render the target recipient cells more susceptible to
transformation by electroporation than untreated cells.
Alternatively, recipient cells are made more susceptible to
transformation, by mechanical wounding.
[0089] To effect transformation by electroporation one may employ
either friable tissues such as a suspension culture of cells, or
embryogenic callus, or alternatively, one may transform immature
embryos or other organized tissues directly. One would partially
degrade the cell walls of the chosen cells by exposing them to
pectin-degrading enzymes (pectolyases) or mechanically wounding in
a controlled manner. Such cells would then be recipient to DNA
transfer by electroporation, which may be carried out at this
stage, and transformed cells then identified by a suitable
selection or screening protocol dependent on the nature of the
newly incorporated DNA.
[0090] (ii) Microprojectile Bombardment
[0091] A further advantageous method for delivering transforming
DNA segments to plant cells is microprojectile bombardment. In this
method, particles may be coated with nucleic acids and delivered
into cells by a propelling force. Exemplary particles include those
comprised of tungsten, gold, platinum, and the like. It is
contemplated that in some instances DNA precipitation onto metal
particles would not be necessary for DNA delivery to a recipient
cell using microprojectile bombardment. However, it is contemplated
that particles may contain DNA rather than be coated with DNA.
Hence it is proposed that DNA-coated particles may increase the
level of DNA delivery via particle bombardment but are not, in and
of themselves, necessary.
[0092] An advantage of microprojectile bombardment, in addition to
it being an effective means of reproducibly stably transforming
monocots, is that neither the isolation of protoplasts (Cristou et
al., 1988) nor the susceptibility to Agrobacterium infection is
required. An illustrative embodiment of a method for delivering DNA
into maize cells by acceleration is a Biolistics Particle Delivery
System, which can be used to propel particles coated with DNA or
cells through a screen, such as a stainless steel or Nytex screen,
onto a filter surface covered with corn cells cultured in
suspension. The screen disperses the particles so that they are not
delivered to the recipient cells in large aggregates. It is
believed that a screen intervening between the projectile apparatus
and the cells to be bombarded reduces the size of projectiles
aggregate and may contribute to a higher frequency of
transformation by reducing damage inflicted on the recipient cells
by projectiles that are too large.
[0093] For the bombardment, cells in suspension are preferably
concentrated on filters or solid culture medium. Alternatively,
immature embryos or other target cells may be arranged on solid
culture medium. The cells to be bombarded are positioned at an
appropriate distance below the macroprojectile stopping plate. If
desired, one or more screens may be positioned between the
acceleration device and the cells to be bombarded. Through the use
of techniques set forth herein one may obtain up to 1000 or more
foci of cells transiently expressing a marker gene. The number of
cells in a focus which express the exogenous gene product 48 hours
post-bombardment often range from 1 to 10 and average 1 to 3.
[0094] In bombardment transformation, one may optimize the
prebombardment culturing conditions and the bombardment parameters
to yield the maximum numbers of stable transformants. Both the
physical and biological parameters for bombardment are important in
this technology. Physical factors are those that involve
manipulating the DNA/microprojectile precipitate or those that
affect the flight and velocity of either the macro- or
microprojectiles. Biological factors include all steps involved in
manipulation of cells before and immediately after bombardment, the
osmotic adjustment of target cells to help alleviate the trauma
associated with bombardment, and also the nature of the
transforming DNA, such as linearized DNA or intact supercoiled
plasmids. It is believed that pre-bombardment manipulations are
especially important for successful transformation of immature
embryos.
[0095] Accordingly, it is contemplated that one may wish to adjust
various of the bombardment parameters in small scale studies to
fully optimize the conditions. One may particularly wish to adjust
physical parameters such as gap distance, flight distance, tissue
distance, and helium pressure. One may also minimize the trauma
reduction factors (TRFs) by modifying conditions which influence
the physiological state of the recipient cells and which may
therefore influence transformation and integration efficiencies.
For example, the osmotic state, tissue hydration and the subculture
stage or cell cycle of the recipient cells may be adjusted for
optimum transformation. Results from such small scale optimization
studies are disclosed herein and the execution of other routine
adjustments will be known to those of skill in the art in light of
the present disclosure.
[0096] III. Recipient Cells for Transformation
[0097] Tissue culture requires media and controlled environments.
"Media" refers to the numerous nutrient mixtures that are used to
grow cells in vitro, that is, outside of the intact living
organism. The medium is usually a suspension of various categories
of ingredients (salts, amino acids, growth regulators, sugars,
buffers) that are required for growth of most cell types. However,
each specific cell type requires a specific range of ingredient
proportions for growth, and an even more specific range of formulas
for optimum growth. Rate of cell growth will also vary among
cultures initiated with the array of media that permit growth of
that cell type.
[0098] Nutrient media is prepared as a liquid, but this may be
solidified by adding the liquid to materials capable of providing a
solid support. Agar is most commonly used for this purpose.
Bactoagar, Hazelton agar, Gelrite, and Gelgro are specific types of
solid support that are suitable for growth of plant cells in tissue
culture.
[0099] Some cell types will grow and divide either in liquid
suspension or on solid media. As disclosed herein, maize cells will
grow in suspension or on solid medium, but regeneration of plants
from suspension cultures requires transfer from liquid to solid
media at some point in development. The type and extent of
differentiation of cells in culture will be affected not only by
the type of media used and by the environment, for example, pH, but
also by whether media is solid or liquid. Table 2 illustrates the
composition of various media useful for creation of recipient cells
and for plant regeneration.
[0100] Recipient cell targets include, but are not limited to,
meristem cells, Type I, Type II, and Type III callus, immature
embryos and gametic cells such as microspores pollen, sperm and egg
cells. It is contemplated that any cell from which a fertile
transgenic plant may be regenerated is useful as a recipient cell.
Type I, Type II, and Type III callus may be initiated from tissue
sources including, but not limited to, immature embryos, seedling
apical meristems, microspores and the such. Those cells which are
capable of proliferating as callus are also recipient cells for
genetic transformation. The present invention provides techniques
for transforming immature embryos followed by initiation of callus
and subsequent regeneration of fertile transgenic plants. Direct
transformation of immature embryos obviates the need for long term
development of recipient cell cultures. Pollen, as well as its
precursor cells, microspores, may be capable of functioning as
recipient cells for genetic transformation, or as vectors to carry
foreign DNA for incorporation during fertilization. Direct pollen
transformation would obviate the need for cell culture.
Meristematic cells (ie., plant cells capable of continual cell
division and characterized by an undifferentiated cytological
appearance, normally found at growing points or tissues in plants
such as root tips, stem apices, lateral buds, etc.) may represent
another type of recipient plant cell. Because of their
undifferentiated growth and capacity for organ differentiation and
totipotency, a single transformed meristematic cell could be
recovered as a whole transformed plant. In fact, it is proposed
that embryogenic suspension cultures may be an in vitro
meristematic cell system, retaining an ability for continued cell
division in an undifferentiated state, controlled by the media
environment.
[0101] Cultured plant cells that can serve as recipient cells for
transforming with desired DNA segments include corn cells, and more
specifically, cells from Zea mays L. Somatic cells are of various
types. Embryogenic cells are one example of somatic cells which may
be induced to regenerate a plant through embryo formation.
Non-embryogenic cells are those which typically will not respond in
such a fashion. An example of non-embryogenic cells are certain
Black Mexican Sweet (BMS) corn cells. These cells have been
transformed by microprojectile bombardment using the neo gene
followed by selection with the aminoglycoside, kanamycin (Klein et
al., 1989). However, this BMS culture was not found to be
regenerable. The development of embryogenic maize calli and
suspension cultures useful in the context of the present invention,
e.g., as recipient cells for transformation, has been described in
U.S. Pat. No. 5,134,074, which is incorporated herein by
reference.
[0102] Certain techniques may be used that enrich recipient cells
within a cell population. For example, Type II callus development,
followed by manual selection and culture of friable, embryogenic
tissue, generally results in an enrichment of recipient cells for
use in, for example, micro-projectile transformation. Suspension
culturing, particularly using the media disclosed herein, may
improve the ratio of recipient to non-recipient cells in any given
population. Manual selection techniques which can be employed to
select recipient cells may include, e.g., assessing cell morphology
and differentiation, or may use various physical or biological
means. Cryopreservation is a possible method of selecting for
recipient cells.
[0103] Manual selection of recipient cells, e.g., by selecting
embryogenic cells from the surface of a Type II callus, is one
means that may be used in an attempt to enrich for recipient cells
prior to culturing (whether cultured on solid media or in
suspension). The preferred cells may be those located at the
surface of a cell cluster, and may further be identifiable by their
lack of differentiation, their size and dense cytoplasm. The
preferred cells will generally be those cells which are less
differentiated, or not yet committed to differentiation. Thus, one
may wish to identify and select those cells which are
cytoplasmically dense, relatively unvacuolated with a high nucleus
to cytoplasm ratio (e.g., determined by cytological observations),
small in size (e.g., 10-20:m), and capable of sustained divisions
and somatic proembryo formation.
[0104] It is proposed that other means for identifying such cells
may also be employed. For example, through the use of dyes, such as
Evan's blue, which are excluded by cells with relatively
non-permeable membranes, such as embryogenic cells, and taken up by
relatively differentiated cells such as root-like cells and snake
cells (so-called due to their snake-like appearance).
[0105] Other possible means of identifying recipient cells include
the use of isozyme markers of embryogenic cells, such as glutamate
dehydrogenase, which can be detected by cytochemical stains (Fransz
et al., 1989). However, it is cautioned that the use of isozyme
markers such as glutamate dehydrogenase may lead to some degree of
false positives from non-embryogenic cells such as rooty cells
which nonetheless have a relatively high metabolic activity.
[0106] (i) Culturing Cells to be Recipients for Transformation
[0107] The inventors believe that the ability to prepare and
cryopreserve cultures of maize cells is important to certain
aspects of the present invention, in that it provides a means for
reproducibly and successfully preparing cells for particle-mediated
transformation, electroporation, or other methods of DNA
introduction. The studies described below set forth techniques
which have been successfully applied by the inventors to generate
transformable and regenerable cultures of maize cells. A variety of
different types of media have been developed by the inventors and
employed in carrying out various aspects of the invention. The
following table, Table 2, sets forth the composition of the media
preferred by the inventors for carrying out these aspects of the
invention.
2TABLE 2 Tissue Culture Media Which are Used for Type II Callus
Development, Development of Suspension Cultures and Regeneration of
Plant Cells (Specifically Maize Cells) OTHER MEDIA BASAL SU-
COMPONENTS** NO. MEDIUM CROSE pH (Amount/L) 7 MS* 2% 6.0 .25 mg
thiamine .5 mg BAP .5 mg NAA Bactoagar 10 MS 2% 6.0 .25 mg thiamine
1 mg BAP 1 mg 2,4-D 400 mg L-proline Bactoagar 19 MS 2% 6.0 .25 mg
thiamine .25 mg BAP .25 mg NAA Bactoagar 20 MS 3% 6.0 .25 mg 1 mg
BAP 1 mg NAA Bactoagar 52 MS 2% 6.0 .25 mg thiamine 1 mg 2,4-D
10.sup.-7 M ABA BACTOAGAR 101 MS 3% 6.0 MS vitamins 100 mg
myo-inositol Bactoagar 142 MS 6% 6.0 MS vitamins 5 mg BAP 0.186 mg
NAA 0.175 mg IAA 0.403 mg 2IP Bactoagar 157 MS 6% 6.0 MS vitamins
100 mg myo-inositol Bactoagar 163 MS 3% 6.0 MS vitamins 3.3 mg
dicamba 100 mg myo-inositol Bactoagar 171 MS 3% 6.0 MS vitamins .25
mg 2,4-D 10 mg BAP 100 mg myo-inositol Bactoagar 173 MS 6% 6.0 MS
vitamins 5 mg BAP .186 mg NAA .175 mg IAA .403 mg 2IP 10.sup.-7 M
ABA 200 mg myo-inositol Bactoagar 177 MS 3% 6.0 MS vitamins .25 mg
2,4-D 10 mg BAP 10.sup.-7 M ABA 100 mg myo-inositol Bactoagar 185
MS -- 5.8 3 mg BAP .04 mg NAA RT vitamins 1.65 mg thiamine 1.38 g
L-proline 20 g sorbitol Bactoagar 189 MS -- 5.8 3 mg BAP .04 mg NAA
.5 mg niacin 800 mg L-asparagine 100 mg casamino acids 20 g
sorbitol 1.4 g L-proline 100 mg myo-inositol Gelgro 201 N6 2% 5.8
N6 vitamins 2 mg L-glycine 1 mg 2,4-D 100 mg casein hydrolysate 2.9
g L-proline Gelgro 205 N6 2% 5.8 N6 vitamins 2 mg L-glycine .5 mg
2,4-D 100 mg casein hydrolysate 2.9 g L-proline Gelgro 209 N6 6%
5.8 N6 vitamins 2 mg L-glycine 100 mg casein hydrolysate 0.69 g
L-proline Bactoagar 210 N6 3% 5.5 N6 vitamins 2 mg 2,4-D 250 mg Ca
pantothenate 100 mg myo-inositol 790 mg L-asparagine 100 mg casein
hydrolpate 1.4 g L-proline Hazelton agar**** 2 mg L-glycine 212 N6
3% 5.5 N6 vitamins 2 mg L-glycine 2 mg 2,4-D 250 mg Ca pantothenate
100 mg myo-inositol 100 mg casein hydrolysate 1.4 g L-proline
Hazelton agar**** 227 N6 2% 5.8 N6 vitamins 2 mg L-glycine 13.2 mg
dicamba 100 mg casein hydrolysate 2.9 g L-proline Gelgro 273 N6 2%
5.8 N6 vitamins 2 mg L-glycine 1 mg 2,4-D 16.9 mg AgNO.sub.3 100 mg
casein hydrolysate 2.9 g L-proline 279 N6 2% 5.8 3.3 mg dicamba 1
mg thiamine .5 mg niacin 800 mg L-asparagine 100 mg casein
hydrolysate 100 mg myoinositol 1.4 g L-proline Gelgro**** 288 N6 3%
3.3 mg dicamba 1 mg thiamine .5 mg niacin .8 g L-asparagine 100 mg
myo-inosital 1.4 g L-proline 100 mg casein hydrolysate 16.9 mg
AgNO.sub.3 Gelgro 401 MS 3% 6.0 3.73 mg Na.sub.2EDTA .25 mg
thiamine 1 mg 2,4-D 2 mg NAA 200 mg casein hydrolysate 500 mg
K.sub.2SO.sub.4 400 mg KH.sub.2PO.sub.4 100 mg myo-inositol 402 MS
3% 6.0 3.73 mg Na.sub.2EDTA .25 mg thiamine 1 mg 2,4-D 200 mg
casein hydrolysate 2.9 g L-proline 500 mg K.sub.2SO.sub.4 400 mg
KH.sub.2PO.sub.4 100 mg myo-inositol 409 MS 3% 6.0 3.73 mg
Na.sub.2EDTA .25 mg thiamine 9.9 mg dicamba 200 mg casein
hydrolysate 2.9 g L-proline 500 mg K.sub.2SO.sub.4 400 mg
KH.sub.2PO.sub.4 100 mg myo-inositol 501 Clark's 2% 5.7 Medium***
607 1/2 .times. MS 3% 5.8 1 mg thiamine 1 mg niacin Gelrite 615 MS
3% 6.0 MS vitamins 6 mg BAP 100 mg myo-inositol Bactoagar 617 1/2
.times. MS 1.5% 6.0 MS vitamins 50 mg myo-inositol Bactoagar 708 N6
2% 5.8 N6 vitamins 2 mg L-glycine 1.5 mg 2,4-D 200 mg casein
hydrolysate 0.69 g L-proline Gelrite 721 N6 2% 5.8 3.3 mg dicamba 1
mg thiamine .5 mg niacin 800 mg L-asparagine 100 mg myo-inositol
100 mg casein hydrolysate 1.4 g L-proline 54.65 g mannitol Gelgro
726 N6 3% 5.8 3.3 mg dicamba .5 mg niacin 1 mg thiamine 800 mg
L-asparagine 100 mg myo-inositol 100 mg casein hydrolysate 1.4 g
L-proline 727 N6 3% 5.8 N6 vitamins 2 mg L-glycine 9.9 mg dicamba
100 mg casein hydrolysate 2.9 g L-proline Gelgro 728 N6 3% 5.8 N6
vitamins 2 mg L-glycine 9.9 mg dicamba 16.9 mg AgNO.sub.3 100 mg
casein hydrolysate 2.9 g L-proline Gelgro 734 N6 2% 5.8 N6 vitamins
2 mg L-glycine 1.5 mg 2,4-D 14 g Fe sequestreene (replaces Fe-EDTA)
200 mg casein hydrolyste 0.69 g L-proline Gelrite 735 N6 2% 5.8 1
mg 2,4-D .5 mg niacin .91 g L-asparagine 100 mg myo-inositol 1 mg
thiamine .5 g MES .75 g MgCl.sub.2 100 mg casein hydrolysate 0.69 g
L-proline Gelgro 2004 N6 3% 5.8 1 mg thiamine 0.5 mg niacin 3.3 mg
dicamba 17 mg AgNO.sub.3 1.4 g L-proline 0.8 g L-asparagine 100 mg
casein hydrolysate 100 mg myo-inositol Gelrite 2008 N6 3% 5.8 1 mg
thiamine 0.5 mg niacin 3.3 mg dicamba 1.4 g L-proline 0.8 g
L-asparagine Gelrite *Basic MS medium described in Murashige and
Skoog (1962). This medium is typically modified by decreasing the
NH.sub.4NO.sub.3 from 1.64 g/l to 1.55 g/l, and omitting the
pyridoxine HCl, nicotinic acid, myo-inositol and glycine. **NAA =
Napthol Acetic Acid IAA = Indole Acetic Acid 2-IP = 2,isopentyl
adenine 2,4-D = 2,4-Dichlorophenoxyacetic Acid BAP = 6-benzyl
aminopurine ABA = abscisic acid ***Basic medium described in Clark
(1982) ****These media may be made with or without solidifying
agent.
[0108] A number of transformable maize cultures have been developed
using the protocols outlined in the following examples. A
compilation of the cultures initiated and tested for
transformability is set forth in Table 3, with the results of the
studies given in the two right-hand columns. The Table indicates
the general selection protocol that was used for each of these
cultures. The numeral designations under "Protocol" represent the
following:
[0109] 1. Tissue (suspension) was plated on filters, bombarded and
then filters were transferred to culture medium. After 2-7 days,
the filters were transferred to selective medium. Approximately 3
weeks after bombardment, tissue was picked from filters as separate
callus clumps onto fresh selective medium.
[0110] 2. As in 1 above, except after bombardment the suspension
was put back into liquid--subjected to liquid selection for 7-14
days and then pipetted at a low density onto fresh selection
plates.
[0111] 3. Callus was bombarded while sitting directly on medium or
on filters. Cells were transferred to selective medium 1-14 days
after particle bombardment. Tissue was transferred on filters 1-3
times at 2 weeks intervals to fresh selective medium. Callus was
then briefly put into liquid to disperse the tissue onto selective
plates at a low density.
[0112] 4. Callus tissue was transferred onto selective plates one
to seven days after DNA introduction. Tissue was subcultured as
small units of callus on selective plates until transformants were
identified.
[0113] The totals demonstrate that 27 of 37 maize cultures were
transformable. Of those cell lines tested 11 out of 20 have
produced fertile plants and 7 are in progress. As this table
indicates, transformable cultures have been produced from ten
different genotypes of maize, including both hybrid and inbred
varieties. These techniques for development of transformable
cultures are important in direct transformation of intact tissues,
such as immature embryos as these techniques rely on the ability to
select transformants in cultured cell systems.
3TABLE 3 Initiated Maize Cultures Fertile Genotype Culture Method
Transformable Plants A188 .times. B73 G(1 .times. 6)92 1 + - G(1
.times. 6)716 1, 2 + + G(1 .times. 6)82 1 + + G(1 .times. 6)98 1 -
NA G(1 .times. 6)99 1 - NA D(1 .times. 6)122#3 2 - NA D(1 .times.
6)114 2 - NA D(1 .times. 6)17#33 2 - NA HB13-3 3 + + HA133-227 2 -
NA G(6 .times. 1)17#25C 3 + + ABT4 4 + + ABT3 4 + + AB60 4 + + AB61
4 + + AB63 4 + + AB80 4 + + AB82 4 + + ABT6 4 + ND AB12 4 + + PH2 4
+ + AB69 4 + - AB44 4 + - AB62 4 + ND A188 .times. B84 G(1 .times.
M)82 1 + - A188 .times. H99 HJ11-7 3 + - B73 .times. A188 G(6
.times. 1)12#7 2 - NA D(6 .times. 1)11#43 2 - NA E1 2 + - Hi-II
G(CW)31#24 + + B73 (6)91#3 2 - NA (6)91#2 2 - NA B73-derived AT824
1, 2, 3 + + N1017A AZ11137a 2 + - Cat 100 CB 2 + ND CC 2 + ND A188
E4 2 + - The symbol "-" indicates that the line was not
transformable after 3 attempts or plants were sterile NA indicates
Not Applicable ND indicates Not Done
[0114] (ii) Media
[0115] In certain embodiments, recipient cells are selected
following growth in culture. Where employed, cultured cells may be
grown either on solid supports or in the form of liquid
suspensions. In either instance, nutrients may be provided to the
cells in the form of media, and environmental conditions
controlled. There are many types of tissue culture media comprised
of amino acids, salts, sugars, growth regulators and vitamins. Most
of the media employed in the practice of the invention will have
some similar components (see, Table 2), the media differ in the
composition and proportions of their ingredients depending on the
particular application envisioned. For example, various cell types
usually grow in more than one type of media, but will exhibit
different growth rates and different morphologies, depending on the
growth media In some media, cells survive but do not divide.
[0116] Various types of media suitable for culture of plant cells
have been previously described. Examples of these media include,
but are not limited to, the N6 medium described by Chu et al.
(1975) and MS media (Murashige & Skoog, 1962). The inventors
have discovered that media such as MS which have a high
ammonia/nitrate ratio are counterproductive to the generation of
recipient cells in that they promote loss of morphogenic capacity.
N6 media, on the other hand, has a somewhat lower ammonia/nitrate
ratio, and is contemplated to promote the generation of recipient
cells by maintaining cells in a proembryonic state capable of
sustained divisions.
[0117] (iii) Maintenance
[0118] The method of maintenance of cell cultures may contribute to
their utility as sources of recipient cells for transformation.
Manual selection of cells for transfer to fresh culture medium,
frequency of transfer to fresh culture medium, composition of
culture medium, and environment factors including, but not limited
to, light quality and quantity and temperature are all important
factors in maintaining callus and/or suspension cultures that are
useful as sources of recipient cells. It is contemplated that
alternating callus between different culture conditions may be
beneficial in enriching for recipient cells within a culture. For
example, it is proposed that cells may be cultured in suspension
culture, but transferred to solid medium at regular intervals.
After a period of growth on solid medium cells can be manually
selected for return to liquid culture medium. It is proposed that
by repeating this sequence of transfers to fresh culture medium it
is possible to enrich for recipient cells. It is also contemplated
that passing cell cultures through a 1.9 mm sieve is useful in
maintaining the friability of a callus or suspension culture and
may be beneficial is enriching for transformable cells.
[0119] (iv) Cryopreservation Methods
[0120] Cryopreservation is important because it allows one to
maintain and preserve a known transformable cell culture for future
use, while eliminating the cumulative detrimental effects
associated with extended culture periods.
[0121] Cell suspensions and callus were cryopreserved using
modifications of methods previously reported (Finkle, 1985; Withers
& King, 1979). The cryopreservation protocol comprised adding a
pre-cooled (0.degree. C.) concentrated cryoprotectant mixture
stepwise over a period of one to two hours to precooled (0.degree.
C.) cells. The mixture was maintained at 0.degree. C. throughout
this period. The volume of added cryoprotectant was equal to the
initial volume of the cell suspension (1:1 addition), and the final
concentration of cryoprotectant additives was 10% dimethyl
sulfoxide, 10% polyethylene glycol (6000 MW), 0.23 M proline and
0.23 M glucose. The mixture was allowed to equilibrate at 0.degree.
C. for 30 minutes, during which time the cell
suspension/cryoprotectant mixture was divided into 1.5 ml aliquot
(0.5 ml packed cell volume) in 2 ml polyethylene cryo-vials. The
tubes were cooled at 0.5.degree. C./minute to -8.degree. C. and
held at this temperature for ice nucleation.
[0122] Once extracellular ice formation had been visually
confirmed, the tubes were cooled at 0.5.degree. C./minute from
-8.degree. C. to -35.degree. C. They were held at this temperature
for 45 minutes (to insure uniform freeze-induced dehydration
throughout the cell clusters). At this point, the cells had lost
the majority of their osmotic volume (i.e. there is little free
water left in the cells), and they could be safely plunged into
liquid nitrogen for storage. The paucity of free water remaining in
the cells in conjunction with the rapid cooling rates from -35 to
-196.degree. C. prevented large organized ice crystals from forming
in the cells. The cells are stored in liquid nitrogen, which
effectively immobilizes the cells and slows metabolic processes to
the point where long-term storage should not be detrimental.
[0123] Thawing of the extracellular solution was accomplished by
removing the cryo-tube from liquid nitrogen and swirling it in
sterile 42.degree. C. water for approximately 2 minutes. The tube
was removed from the heat immediately after the last ice crystals
had melted to prevent heating the tissue. The cell suspension
(still in the cryoprotectant mixture) was pipetted onto a filter,
resting on a layer of BMS cells (the feeder layer which provided a
nurse effect during recovery). Dilution of the cryoprotectant
occurred slowly as the solutes diffused away through the filter and
nutrients diffused upward to the recovering cells. Once subsequent
growth of the thawed cells was noted, the growing tissue was
transferred to fresh culture medium. The cell clusters were
transferred back into liquid suspension medium as soon as
sufficient cell mass had been regained (usually within 1 to 2
weeks). After the culture was reestablished in liquid (within 1 to
2 additional weeks), it was used for transformation experiments.
When desired, previously cryopreserved cultures may be frozen again
for storage.
[0124] IV. DNA Segments Comprising Exogenous Genes
[0125] As mentioned previously, there are several methods to
construct the DNA segments carrying DNA into a host cell that are
well known to those skilled in the art. The general construct of
the vectors used herein are plasmids comprising a promoter, other
regulatory regions, structural genes, and a 3' end.
[0126] The plants of the current invention have a mutant EPSPS gene
which confers glyphosate resistance. The preferred EPSPS sequence,
as shown in FIG. 12, includes a chloroplast transit peptide from
maize in combination with the EPSPS gene. It is to be understood,
that this chloroplast transit peptide could be homologous, i.e.,
from the maize EPSPS gene, or heterologous, i.e., from any other
gene. Preferably the transit peptide will be the optimized transit
peptide used in the constructs disclosed herein. Alternatively, the
EPSPS gene may be used without a transit peptide and the gene
transformed into the chloroplast genome following the techniques
described in U.S. Pat. No. 5,451,513, specifically incorporated
herein by reference.
[0127] Several plasmids encoding a variety of different genes have
been constructed by the present inventors, the important features
of which are represented below in Table 4. Certain of these
plasmids are also shown in FIGS. 1-4: pDPG165 (FIG. 1), pDPG427
(FIG. 2), pDPG434 (FIG. 3), and pDPG443 (FIG. 4).
[0128] Table 4 shows vectors used in the construction of maize
glyphosate resistant lines GA21, GG25, GJ11, and FI117. Table 5
shows the components of the plasmid pDPG434, which was used in the
transformation of GA21 and FI117. The gene encoding the enzyme
EPSPS was cloned from Zea mays. Two mutations were introduced into
the amino acid sequence of EPSPS to confer glyphosate resistance,
i.e., a substitution of isoleucine for threonine at amino acid
position 102 and a substitution of serine for proline at amino acid
position 106. Plant expression vectors pDPG427, pDPG 434, and
pDPG443 were constructed using the promoterless mutant maize EPSPS
expression vector obtained from Rhone Poulenc Agrochimie (pDPG425).
The mutant EPSPS gene in this vector encodes an enzyme with amino
acid changes at positions 102 (threonine to isoleucine) and 106
(proline to serine). A description of the construction of these
vectors is presented herein.
4TABLE 4 Vectors used in the transformation of maize glyphosate
resistant lines GA21, GG25, GJ11, and FI117 RECOMBINANT VECTOR
DELIBERATE DESIGNATION PARENT EXPRESSION & SOURCE REPLICON
INSERT DNA ATTEMPT pDPG165 pUC19 1, 3, 4 1 pDPG427 pSK- 2, 5, 6, 7
2 pDPG434 pSK- 2, 9, 7, 6 2, 7 pDPG443 pSK- 2, 6, 7, 8 2, 7
[0129] KEY: Insert DNA and Deliberate Expression Attempt
[0130] 1. The bar gene from Streptomyces hygroscopicus encodes
phosphinothricin acetyltransferase (PAT). Cells expressing PAT are
resistant to the herbicide Basta. White, J., Chang, S.-Y. P., Bibb,
M. J., and Bibb, M. J. 1990. Nucl. Ac. Research 18: 1062.
[0131] 2. The EPSPS gene (5-enolpyruvy/shikimate-3-phosphate
synthase) gene from Zea Mays was mutated to confer resistance to
the herbicide glyphosate. An isoleucine has been substituted for
threonine at amino acid position 102 and a serine has been
substituted for proline at amino acid position 106.
[0132] 3. Promoter sequences from the Cauliflower Mosaic Virus
genome. Odell, J. T., Nagy, F., and Chua, N.-H. 1985. Nature313:
810-812.
[0133] 4. Terminator sequence from the Ti plasmid of Agrobacterium
tumefaciens. (a) Bevan, M., 1984. Nucleic Acid Research 12:
8711-8721; (b) Ingelbrecht, I. L. W., Herman, L. M. F., DeKeyser,
R. A., Van Montagu, M. C., Depicker, A. G. 1989. The Plant Cell 1:
671-680; (c) Bevan, M., Barnes, W. M., Chilton, M. D., 1983.
Nucleic Acid Research. 11: 369-385; (d) Ellis, J. G., Llewellyn, D.
J., Walker, J. C., Dennis, E. S., Peacocu, W J. 1987. EMBO J. 6:
3203-3208.
[0134] 5. Enhancer sequences from the maize alcohol dehydrogenase
gene. Callis, J., Fromm, M. E., Walbot, V., 1987. Genes Dev. 1:
1183-1200.
[0135] 6. Terminator sequences from Ti plasmid of Agrobacterium
(nos 3'-end) (a) Bevan, M., 1984. Nucleic Acid Research 12:
8711-8721; (b) Ingelbrecht, I. L. W., Herman, L. M. F., DeKeyser,
R. A., Van Montagu, M. C., Depicker, A. G. 1989. The Plant Cell 1:
671-680; (c) Bevan, M., Barnes, W. M., Chilton, M. D., 1983.
Nucleic Acid Research. 11: 369-385.
[0136] 7. A chloroplast transit peptide sequence, referred to here
as the optimized transit peptide sequence (OTP), consisting of DNA
sequence from maize and sunflower ribulose-1,5-bis phosphate
carboxylase oxygenase (RuBisCo) genes (Lebrun et al., 1996; Rhone
Poulenc Agrochimie).
[0137] 8. Fused promoter sequences from Cauliflower Mosaic Virus
genome and Arabidopsis thaliana H4 histone gene. Constructed by
Rhone Poulenc Agrochimie.
[0138] 9. Actin-1 5' region including promoter from Oryza sativa
(McElroy et al. 1991).
5TABLE 5 Summary of Sequences Present in Plasmid pDPG434 Vector
Approx. Component Size, Kb Description rice actin 1.37 5' region of
the rice actin 1 promoter and gene containing the promoter and
intron first intron (McElroy et al., 1991) optimized 0.37
chloroplast transit peptide transit peptide sequence constructed
based on (OTP) transit peptide sequences from maize and sunflower
ribulose-1,5- bis phosphate carboxylase oxygenase (RuBisCo) genes
(Lebrun et al., 1996) mutant maize 1.34 wild-type maize EPSPS gene
EPSPS gene (Lebrun et al., 1991) containing mutations at amino acid
position 102 (threonine to isoleucine) and 106 (proline to serine)
nos 3'-end 0.24 polyadenlylation region from the nopaline synthase
gene from Agrobacterium tumefaciens (Bevan, 1984) lac 0.24 A
partial lacI coding sequence, the promoter plac, and a partial
coding sequence for .beta.-galactosidase or lacZ protein
(Yanisch-Perron et al., 1985) bla 0.86 The TEM type
.beta.-lactamase gene from E. coli plasmid pBR322 confers
resistance on bacterial cells to ampicillin and other penicillins
(Sutcliffe, 1978). The gene is under control of its native
bacterial promoter. ColE1 ori 0.65 The origin of DNA replication
from the E. coli high copy plasmid pUC19 (Yanisch-Perron et al.,
1985)
[0139] V. Identification of Transformed Cells Using Selection
[0140] It is believed that DNA is introduced into only a small
percentage of cells in any one experiment. In order to provide a
more efficient system for identification of those cells receiving
DNA and integrating it into their genomes, therefore, one may
desire to employ a means for selecting those cells that are stably
transformed. One exemplary embodiment of such a method is to
introduce into the host cell, a marker gene which confers
resistance to some normally inhibitory agent, e.g. an antibiotic or
herbicide. The potentially transformed cells are then exposed to
the agent. In the population of surviving cells are those cells
wherein generally the resistance-conferring gene has been
integrated and expressed at sufficient levels to permit cell
survival. Cells may be tested further to confirm stable integration
of the exogenous DNA. Using embryogenic suspension cultures, stable
transformants are recovered at a frequency of approximately 1 per
1000 transiently expressing foci.
[0141] One herbicide which has been suggested as a desirable
selection agent is the broad spectrum herbicide bialaphos.
Bialaphos is a tripeptide antibiotic produced by Streptomyces
hygroscopicus and is composed of phosphinothricin (PPT), an
analogue of L-glutamic acid, and two L-alanine residues. Upon
removal of the L-alanine residues by intracellular peptidases, the
PPT is released and is a potent inhibitor of glutamine synthetase
(GS), a pivotal enzyme involved in ammonia assimilation and
nitrogen metabolism (Ogawa et al., 1973). Synthetic PPT, the active
ingredient in the herbicide Liberty.TM. is also effective as a
selection agent. Inhibition of GS in plants by PPT causes the rapid
accumulation of ammonia and death of the plant cells.
[0142] The organism producing bialaphos and other species of the
genus Streptomyces also synthesizes an enzyme phosphinothricin
acetyl transferase (PAT) which is encoded by the bar gene in
Streptomyces hygroscopicus and the pat gene in Streptomyces
viridochromogenes. The use of the herbicide resistance gene
encoding phosphinothricin acetyl transferase (PAT) is referred to
in DE 3642 829 A, wherein the gene is isolated from Streptomyces
viridochromogenes. In the bacterial source organism this enzyme
acetylates the free amino group of PPT preventing auto-toxicity
(Thompson et al., 1987). The bar gene has been cloned (Murakami et
al., 1986; Thompson et al., 1987) and expressed in transgenic
tobacco, tomato and potato plants (De Block, 1987) and Brassica (De
Block, 1989). In previous reports, some transgenic plants which
expressed the resistance gene were completely resistant to
commercial formulations of PPT and bialaphos in greenhouses.
[0143] Another herbicide which is useful for selection of
transformed cell lines in the practice of the invention is the
broad spectrum herbicide glyphosate. Glyphosate inhibits the action
of the enzyme EPSPS which is active in the aromatic amino acid
biosynthetic pathway. Inhibition of this enzyme leads to starvation
for the amino acids phenylalanine, tyrosine, and tryptophan and
secondary metabolites derived thereof. U.S. Pat. No. 4,535,060
describes the isolation of EPSPS mutations which infer glyphosate
resistance on the Salmonella typhimurium gene for EPSPS, aroA. The
EPSPS gene was cloned from Zea mays and mutations similar to those
found in a glyphosate resistant aroA gene were introduced in vitro.
The mutant gene encodes a protein with amino acid changes at
residues 102 and 106. Although these mutations confer resistance to
glyphosate on the enzyme EPSPS it is anticipated that other
mutations will also be useful.
[0144] Exemplary embodiments of vectors capable of delivering DNA
to plant host cells in the current invention are the plasmids,
pDPG165, pDPG427, pDPG434, and pDPG443. These and other suitable
plasmid vectors are further discussed in U.S. patent application
Ser. No. 08/113,561, filed Aug. 25, 1993, which is specifically
incorporated herein by reference. A very important component of the
pDPG165 plasmid for purposes of genetic transformation is the bar
gene which encodes a marker for selection of transformed cells
exposed to bialaphos or PPT. Plasmids pDPG434, pDPG427, pDPG441,
pDPG443, and pDPG436, pDPG447, pDPG465, and pDPG467 contain a maize
EPSPS gene with mutations at amino acid residues 102 and 106 driven
by various different promoters (U.S. patent application Ser. No.
08/113,561, filed Aug. 25, 1993). A very important component of
these plasmids for purposes of genetic transformation is the
mutated EPSPS gene which encodes a marker for selection of
transformed cells.
[0145] VI. Production and Characterization of Stable Transgenic
Corn
[0146] After effecting delivery of exogenous DNA to recipient
cells, the next steps generally concern identifying the transformed
cells for further culturing and plant regeneration. As mentioned
herein, in order to improve the ability to identify transformants,
one may desire to employ a selectable or screenable marker gene as,
or in addition to, the expressible gene of interest. In this case,
one would then generally assay the potentially transformed cell
population by exposing the cells to a selective agent or agents, or
one would screen the cells for the desired marker gene trait.
[0147] (i) Selection
[0148] An exemplary embodiment of methods for identifying
transformed cells involves exposing the bombarded cultures to a
selective agent, such as a metabolic inhibitor, an antibiotic,
herbicide or the like. Cells which have been transformed and have
stably integrated a marker gene conferring resistance to the
selective agent used, will grow and divide in culture. Sensitive
cells will not be amenable to further culturing.
[0149] To use the bar-bialaphos or the EPSPS-glyphosate selective
system, bombarded tissue is cultured for 0-28 days on nonselective
medium and subsequently transferred to medium containing from 1-3
mg/l bialaphos or 1-3 nM glyphosate as appropriate. While ranges of
1-3 mg/l bialaphos or 1-3 mM glyphosate will typically be
preferred, it is proposed that ranges of 0.1-50 mg/l bialaphos or
0.1-50 mM glyphosate will find utility in the practice of the
invention. Tissue can be placed on any porous, inert, solid or
semi-solid support for bombardment, including but not limited to
filters and solid culture medium. Bialaphos and glyphosate are
provided as examples of agents suitable for selection of
transformants, but the technique of this invention is not limited
to them.
[0150] An example of a screenable marker trait is the red pigment
produced under the control of the R-locus in maize. This pigment
may be detected by culturing cells on a solid support containing
nutrient media capable of supporting growth at this stage and
selecting cells from colonies (visible aggregates of cells) that
are pigmented. These cells may be cultured further, either in
suspension or on solid media. The R-locus is useful for selection
of transformants from bombarded immature embryos. In a similar
fashion, the introduction of the C1 and B genes will result in
pigmented cells and/or tissues.
[0151] The enzyme luciferase may be used as a screenable marker in
the context of the present invention. In the presence of the
substrate luciferin, cells expressing luciferase emit light which
can be detected on photographic or x-ray film, in a luminometer (or
liquid scintillation counter), by devices that enhance night
vision, or by a highly light sensitive video camera, such as a
photon counting camera. All of these assays are nondestructive and
transformed cells may be cultured further following identification.
The photon counting camera is especially valuable as it allows one
to identify specific cells or groups of cells which are expressing
luciferase and manipulate those in real time. Another screenable
marker which may be used is the gene coding for green fluorescent
protein.
[0152] It is further contemplated that combinations of screenable
and selectable markers will be useful for identification of
transformed cells. In some cell or tissue types a selection agent,
such as bialaphos or glyphosate, may either not provide enough
killing activity to clearly recognize transformed cells or may
cause substantial nonselective inhibition of transformants and
nontransformants alike, thus causing the selection technique to not
be effective. It is proposed that selection with a growth
inhibiting compound, such as bialaphos or glyphosate at
concentrations below those that cause 100% inhibition followed by
screening of growing tissue for expression of a screenable marker
gene such as luciferase would allow one to recover transformants
from cell or tissue types that are not amenable to selection alone.
It is proposed that combinations of selection and screening may
enable one to identify transformants in a wider variety of cell and
tissue types.
[0153] (ii) Regeneration and Seed Production
[0154] Cells that survive the exposure to the selective agent, or
cells that have been scored positive in a screening assay, may be
cultured in media that supports regeneration of plants. In an
exemplary embodiment, MS and N6 media may be modified (see Table 2)
by including further substances such as growth regulators. A
preferred growth regulator for such purposes is dicamba or 2,4-D.
However, other growth regulators may be employed, including NAA,
NAA+2,4-D or perhaps even picloram. Media improvement in these and
like ways has been found to facilitate the growth of cells at
specific developmental stages. Tissue may be maintained on a basic
media with growth regulators until sufficient tissue is available
to begin plant regeneration efforts, or following repeated rounds
of manual selection, until the morphology of the tissue is suitable
for regeneration, at least two weeks, then transferred to media
coiducive to maturation of embryoids. Cultures are transferred
every two weeks on this medium. Shoot development will signal the
time to transfer to medium lacking growth regulators.
[0155] The transformed cells, identified by selection or screening
and cultured in an appropriate medium that supports regeneration,
will then be allowed to mature into plants. Developing plantlets
are transferred to soiless plant growth mix, and hardened, e.g., in
an environmentally controlled chamber at about 85% relative
humidity, 600 ppm CO.sub.2, and 25-250 microeinsteins
m.sup.-2s.sup.-1 of light. Plants are preferably matured either in
a growth chamber or greenhouse. Plants are regenerated from about 6
weeks to 10 months after a transformant is identified, depending on
the initial tissue. During regeneration, cells are grown on solid
media in tissue culture vessels. Illustrative embodiments of such
vessels are petri dishes and Plant Cons. Regenerating plants are
preferably grown at about 19 to 28.degree. C. After the
regenerating plants have reached the stage of shoot and root
development, they may be transferred to a greenhouse for further
growth and testing.
[0156] Note, however, that kernels on transformed plants may
occasionally require embryo rescue due to cessation of kernel
development and premature senescence of plants. To rescue
developing embryos, they are excised from surface-disinfected
kernels 10-20 days post-pollination and cultured. An embodiment of
media used for culture at this stage comprises MS salts, 2%
sucrose, and 5.5 g/l agarose. In embryo rescue, large embryos
(defined as greater than 3 mm in length) are germinated directly on
an appropriate media. Embryos smaller than that may be cultured for
one week on media containing the above ingredients along with
10.sup.-5M abscisic acid and then transferred to growth
regulator-free medium for germination.
[0157] Progeny may be recovered from the transformed plants and
tested for expression of the exogenous expressible gene by
localized application of an appropriate substrate to plant parts
such as leaves. In the case of bar transformed plants, it was found
that transformed parental plants (R.sub.o) and their progeny
(R.sub.1) exhibited no bialaphos-related necrosis after localized
application of the herbicide Basta7 to leaves, if there was
functional PAT activity in the plants as assessed by an in vitro
enzymatic assay. All PAT positive progeny tested contained bar,
confirming that the presence of the enzyme and the resistance to
bialaphos were associated with the transmission through the
germline of the marker gene.
[0158] (iii) Characterization
[0159] To confirm the presence of the exogenous DNA or "transgene
(s)" in the regenerating plants, a variety of assays may be
performed. Such assays include, for example, "molecular biological"
assays, such as Southern and Northern blotting and PCR;
"biochemical" assays, such as detecting the presence of a protein
product, e.g., by immunological means (ELISAs and Western blots) or
by enzymatic function; plant part assays, such as leaf or root
assays; and also, by analyzing the phenotype of the whole
regenerated plant.
[0160] 1. DNA Integration, RNA Expression and Inheritance
[0161] Genomic DNA may be isolated from callus cell lines or any
plant parts to determine the presence of the exogenous gene through
the use of techniques well known to those skilled in the art. Note,
that intact sequences will not always be present, presumably due to
rearrangement or deletion of sequences in the cell.
[0162] The presence of DNA elements introduced through the methods
of this invention may be determined by polymerase chain reaction
(PCR). Using this technique discreet fragments of DNA are amplified
and detected by gel electrophoresis. This type of analysis permits
one to determine whether a gene is present in a stable
transformant, but does not prove integration of the introduced gene
into the host cell genome. It is the experience of the inventors,
however, that DNA has been integrated into the genome of all
transformants that demonstrate the presence of the gene through PCR
analysis. In addition, it is not possible using PCR techniques to
determine whether transformants have exogenous genes introduced
into different sites in the genome, i.e., whether transformants are
of independent origin. It is contemplated that using PCR techniques
it would be possible to clone fragments of the host genomic DNA
adjacent to an introduced gene.
[0163] Positive proof of DNA integration into the host genome and
the independent identities of transformants may be determined using
the technique of Southern hybridization. Using this technique
specific DNA sequences that were introduced into the host genome
and flanking host DNA sequences can be identified. Hence the
Southern hybridization pattern of a given transformant serves as an
identifying characteristic of that transformant. In addition it is
possible through Southern hybridization to demonstrate the presence
of introduced genes in high molecular weight DNA, i.e., confirm
that the introduced gene has been integrated into the host cell
genome. The technique of Southern hybridization provides
information that is obtained using PCR, e.g., the presence of a
gene, but also demonstrates integration into the genome and
characterizes each individual transformant.
[0164] It is contemplated that using the techniques of dot or slot
blot hybridization which are modifications of Southern
hybridization techniques one could obtain the same information that
is derived from PCR, e.g., the presence of a gene.
[0165] Both PCR and Southern hybridization techniques can be used
to demonstrate transmission of a transgene to progeny. In most
instances the characteristic Southern hybridization pattern for a
given transformant will segregate in progeny as one or more
Mendelian genes (Spencer et al., 1992) indicating stable
inheritance of the transgene.
[0166] Whereas DNA analysis techniques may be conducted using DNA
isolated from any part of a plant, RNA will only be expressed in
particular cells or tissue types and hence it will be necessary to
prepare RNA for analysis from these tissues. PCR techniques may
also be used for detection and quantitation of RNA produced from
introduced genes. In this application of PCR it is first necessary
to reverse transcribe RNA into DNA, using enzymes such as reverse
transcriptase, and then through the use of conventional PCR
techniques amplify the DNA. In most instances PCR techniques, while
useful, will not demonstrate integrity of the RNA product. Further
information about the nature of the RNA product may be obtained by
Northern blotting. This technique will demonstrate the presence of
an RNA species and give information about the integrity of that
RNA. The presence or absence of an RNA species can also be
determined using dot or slot blot Northern hybridizations. These
techniques are modifications of Northern blotting and will only
demonstrate the presence or absence of an RNA species.
[0167] 2. Gene Expression
[0168] While Southern blotting and PCR may be used to detect the
gene(s) in question, they do not provide information as to whether
the gene is being expressed. Expression may be evaluated by
specifically identifying the protein products of the introduced
genes or evaluating the phenotypic changes brought about by their
expression.
[0169] Assays for the production and identification of specific
proteins may make use of physical-chemical, structural, functional,
or other properties of the proteins. Unique physical-chemical or
structural properties allow the proteins to be separated and
identified by electrophoretic procedures, such as native or
denaturing gel electrophoresis or isoelectric focusing, or by
chromatographic techniques such as ion exchange or gel exclusion
chromatography. The unique structures of individual proteins offer
opportunities for use of specific antibodies to detect their
presence in formats such as an ELISA assay. Combinations of
approaches may be employed with even greater specificity such as
western blotting in which antibodies are used to locate individual
gene products that have been separated by electrophoretic
techniques. Additional techniques may be employed to absolutely
confirm the identity of the product of interest such as evaluation
by amino acid sequencing following purification. Although these are
among the most commonly employed, other procedures may be
additionally used.
[0170] Assay procedures also may be used to identify the expression
of proteins by their functionality, especially the ability of
enzymes to catalyze specific chemical reactions involving specific
substrates and products. These reactions may be followed by
providing and quantifying the loss of substrates or the generation
of products of the reactions by physical or chemical procedures.
Examples are as varied as the enzyme to be analyzed and may include
assays for PAT enzymatic activity by following production of
radiolabeled acetylated phosphinothricin from phosphinothricin and
.sup.14C-acetyl CoA or for anthranilate synthase activity by
following loss of fluorescence of anthranilate, to name two.
[0171] Very frequently the expression of a gene product is
determined by evaluating the phenotypic results of its expression.
These assays also may take many forms including but not limited to
analyzing changes in the chemical composition, morphology, or
physiological properties of the plant. Chemical composition may be
altered by expression of genes encoding enzymes or storage proteins
which change amino acid composition and may be detected by amino
acid analysis, or by enzymes which change starch quantity which may
be analyzed by near infrared reflectance spectrometry.
Morphological changes may include greater stature or thicker
stalks. Most often changes in response of plants or plant parts to
imposed treatments are evaluated under carefully controlled
conditions termed bioassays.
[0172] VII. Purification of Proteins
[0173] It may, in particular embodiments of the current invention,
be desirable to purify proteins encoded by transgenes of the
current invention. Protein purification techniques are well known
to those of skill in the art. These techniques involve, at one
level, the crude fractionation of the cellular milieu to
polypeptide and non-polypeptide fractions. Having separated the
polypeptide from other proteins, the polypeptide of interest may be
further purified using chromatographic and electrophoretic
techniques to achieve partial or complete purification (or
purification to homogeneity). Analytical methods particularly
suited to the preparation of a pure peptide are ion-exchange
chromatography, exclusion chromatography; polyacrylamide gel
electrophoresis; and isoelectric focusing. A particularly efficient
method of purifying peptides is fast protein liquid chromatography
or even HPLC.
[0174] Certain aspects of the present invention concern the
purification, and in particular embodiments, the substantial
purification, of an encoded protein or peptide. The term "purified
protein or peptide" as used herein, is intended to refer to a
composition, isolatable from other components, wherein the protein
or peptide is purified to any degree relative to its
naturally-obtainable state. A purified protein or peptide therefore
also refers to a protein or peptide, free from the environment in
which it may naturally occur.
[0175] Generally, "purified" will refer to a protein or peptide
composition that has been subjected to fractionation to remove
various other components, and which composition substantially
retains its expressed biological activity. Where the term
"substantially purified" is used, this designation will refer to a
composition in which the protein or peptide forms the major
component of the composition, such as constituting about 50%, about
60%, about 70%, about 80%, about 90%, about 95% or more of the
proteins in the composition.
[0176] Various methods for quantifying the degree of purification
of the protein or peptide will be known to those of skill in the
art in light of the present disclosure. These include, for example,
determining the specific activity of an active fraction, or
assessing the amount of polypeptides within a fraction by SDS/PAGE
analysis. A preferred method for assessing the purity of a fraction
is to calculate the specific activity of the fraction, to compare
it to the specific activity of the initial extract, and to thus
calculate the degree of purity, herein assessed by a "-fold
purification number." The actual units used to represent the amount
of activity will, of course, be dependent upon the particular assay
technique chosen to follow the purification and whether or not the
expressed protein or peptide exhibits a detectable activity.
[0177] Various techniques suitable for use in protein purification
will be well known to those of skill in the art. These include, for
example, precipitation with ammonium sulphate, PEG, antibodies and
the like or by heat denaturation, followed by centrifugation;
chromatography steps such as ion exchange, gel filtration, reverse
phase, hydroxylapatite and affinity chromatography; isoelectric
focusing; gel electrophoresis; and combinations of such and other
techniques. As is generally known in the art, it is believed that
the order of conducting the various purification steps may be
changed, or that certain steps may be omitted, and still result in
a suitable method for the preparation of a substantially purified
protein or peptide.
[0178] There is no general requirement that the protein or peptide
always be provided in their most purified state. Indeed, it is
contemplated that less substantially purified products will have
utility in certain embodiments. Partial purification may be
accomplished by using fewer purification steps in combination, or
by utilizing different forms of the same general purification
scheme. For example, it is appreciated that a cation-exchange
column chromatography performed utilizing an HPLC apparatus will
generally result in a greater "-fold" purification than the same
technique utilizing a low pressure chromatography system. Methods
exhibiting a lower degree of relative purification may have
advantages in total recovery of protein product, or in maintaining
the activity of an expressed protein.
[0179] It is known that the migration of a polypeptide can vary,
sometimes significantly, with different conditions of SDS/PAGE
(Capaldi et al., 1977). It will therefore be appreciated that under
differing electrophoresis conditions, the apparent molecular
weights of purified or partially purified expression products may
vary.
[0180] High Performance Liquid Chromatography (HPLC) is
characterized by a very rapid separation with extraordinary
resolution of peaks. This is achieved by the use of very fine
particles and high pressure to maintain an adequate flow rate.
Separation can be accomplished in a matter of minutes, or at most
an hour. Moreover, only a very small volume of the sample is needed
because the particles are so small and close-packed that the void
volume is a very small fraction of the bed volume. Also, the
concentration of the sample need not be very great because the
bands are so narrow that there is very little dilution of the
sample.
[0181] Gel chromatography, or molecular sieve chromatography, is a
special type of partition chromatography that is based on molecular
size. The theory behind gel chromatography is that the column,
which is prepared with tiny particles of an inert substance that
contain small pores, separates larger molecules from smaller
molecules as they pass through or around the pores, depending on
their size. As long as the material of which the particles are made
does not adsorb the molecules, the sole factor determining rate of
flow is the size. Hence, molecules are eluted from the column in
decreasing size, so long as the shape is relatively constant. Gel
chromatography is unsurpassed for separating molecules of different
size because separation is independent of all other factors such as
pH, ionic strength, temperature, etc. There also is virtually no
adsorption, less zone spreading and the elution volume is related
in a simple matter to molecular weight.
[0182] Affinity Chromatography is a chromatographic procedure that
relies on the specific affinity between a substance to be isolated
and a molecule that it can specifically bind to. This is a
receptor-ligand type interaction. The column material is
synthesized by covalently coupling one of the binding partners to
an insoluble matrix. The column material is then able to
specifically adsorb the substance from the solution. Elution occurs
by changing the conditions to those in which binding will not occur
(alter pH, ionic strength, temperature, etc.).
[0183] A particular type of affinity chromatography useful in the
purification of carbohydrate containing compounds is lectin
affinity chromatography. Lectins are a class of substances that
bind to a variety of polysaccharides and glycoproteins. Lectins are
usually coupled to agarose by cyanogen bromide. Conconavalin A
coupled to Sepharose was the first material of this sort to be used
and has been widely used in the isolation of polysaccharides and
glycoproteins other lectins that have been include lentil lectin,
wheat germ agglutinin which has been useful in the purification of
N-acetyl glucosaminyl residues and Helix pomatia lectin. Lectins
themselves are purified using affinity chromatography with
carbohydrate ligands. Lactose has been used to purify lectins from
castor bean and peanuts; maltose has been useful in extracting
lectins from lentils and jack bean; N-acetyl-D galactosamine is
used for purifying lectins from soybean; N-acetyl glucosaminyl
binds to lectins from wheat germ; D-galactosamine has been used in
obtaining lectins from clams and L-fucose will bind to lectins from
lotus.
[0184] The matrix should be a substance that itself does not adsorb
molecules to any significant extent and that has a broad range of
chemical, physical and thermal stability. The ligand should be
coupled in such a way as to not affect its binding properties. The
ligand should also provide relatively tight binding. And it should
be possible to elute the substance without destroying the sample or
the ligand. One of the most common forms of affinity chromatography
is immunoaffinity chromatography. The generation of antibodies that
would be suitable for use in accord with the present invention is
discussed below.
[0185] VIII. Genetic Analysis of Glyphosate Resistant Transgenic
Plants
[0186] In particular embodiments of the invention, methods may be
used for detecting variation in the expression of particular
transgenes such as the bar gene and mutant EPSPS. This method may
comprise determining the level of protein expressed by these genes
or by determining specific alterations in the expressed product.
Obviously, this sort of assay has importance in the screening of
transformants for potential herbicide resistance. Such assays may
in some cases be faster, more accurate or less expensive than
conventional screening assays.
[0187] The biological sample may potentially be any type of plant
tissue. Nucleic acid is isolated from cells contained in the
biological sample, according to standard methodologies (Sambrook et
al., 1989). The nucleic acid may be genomic DNA or fractionated or
whole cell RNA. Where RNA is used, it may be desired to convert the
RNA to a complementary DNA. In one embodiment, the RNA is whole
cell RNA; in another, it is poly-A RNA. Normally, the nucleic acid
is amplified.
[0188] Depending on the format, the specific nucleic acid of
interest is identified in the sample directly using amplification
or with a second, known nucleic acid following amplification. Next,
the identified product is detected. In certain applications, the
detection may be performed by visual means (e.g., ethidium bromide
staining of a gel). Alternatively, the detection may involve
indirect identification of the product via chemiluminescence,
radioactive scintigraphy of radiolabel or fluorescent label or even
via a system using electrical or thermal impulse signals (Affymax
Technology; Bellus, 1994).
[0189] Following detection, one may compare the results seen in a
given plant with a statistically significant reference group of
non-transformed control plants. Typically, the non-transformed
control plants will be of a genetic background similar to the
transformed plants. In this way, it is possible to detect
differences in the amount or kind of protein detected in various
transformed plants.
[0190] A variety of different assays are contemplated in the
screening of the glyphosate resistant plants of the current
invention and associated exogenous elements. These techniques may
in cases be used to detect for both the presence of the particular
genes as well as rearrangements that may have occurred in the gene
construct. The techniques include but are not limited to,
fluorescent in situ hybridization (FISH), direct DNA sequencing,
PFGE analysis, Southern or Northern blotting, single-stranded
conformation analysis (SSCA), RNAse protection assay,
allele-specific oligonucleotide (ASO), dot blot analysis,
denaturing gradient gel electrophoresis, RFLP and PCR-SSCP.
[0191] (i) Primers and Probes
[0192] The term primer, as defined herein, is meant to encompass
any nucleic acid that is capable of priming the synthesis of a
nascent nucleic acid in a template-dependent process. Typically,
primers are oligonucleotides from ten to twenty base pairs in
length, but longer sequences can be employed. Primers may be
provided in double-stranded or single-stranded form, although the
single-stranded form is preferred. Probes are defined differently,
although they may act as primers. Probes, while perhaps capable of
priming, are designed to binding to the target DNA or RNA and need
not be used in an amplification process.
[0193] In preferred embodiments, the probes or primers are labeled
with radioactive species (.sup.32P, .sup.14C, .sup.35S, .sup.3H, or
other label), with a fluorophore (rhodamine, fluorescein), an
antigen (biotin, streptavidin, digoxigenin), or a chemillumiscent
(luciferase).
[0194] (ii) Template Dependent Amplification Methods
[0195] A number of template dependent processes are available to
amplify the marker sequences present in a given template sample.
One of the best known amplification methods is the polymerase chain
reaction (referred to as PCR.TM.) which is described in detail in
U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, each of which is
incorporated herein by reference in its entirety.
[0196] Briefly, in PCR, two primer sequences are prepared that are
complementary to regions on opposite complementary strands of the
marker sequence. An excess of deoxynucleoside triphosphates are
added to a reaction mixture along with a DNA polymerase, e.g., Taq
polymerase. If the marker sequence is present in a sample, the
primers will bind to the marker and the polymerase will cause the
primers to be extended along the marker sequence by adding on
nucleotides. By raising and lowering the temperature of the
reaction mixture, the extended primers will dissociate from the
marker to form reaction products, excess primers will bind to the
marker and to the reaction products and the process is
repeated.
[0197] A reverse transcriptase PCR amplification procedure may be
performed in order to quantify the amount of mRNA amplified.
Methods of reverse transcribing RNA into cDNA are well known and
described in Sambrook et al., 1989. Alternative methods for reverse
transcription utilize thermostable, RNA-dependent DNA polymerases.
These methods are described in WO 90/07641 filed Dec. 21, 1990.
Polymerase chain reaction methodologies are well known in the
art.
[0198] Another method for amplification is the ligase chain
reaction ("LCR"), disclosed in EPO No. 320 308, incorporated herein
by reference in its entirety. In LCR, two complementary probe pairs
are prepared, and in the presence of the target sequence, each pair
will bind to opposite complementary strands of the target such that
they abut. In the presence of a ligase, the two probe pairs will
link to form a single unit. By temperature cycling, as in PCR,
bound ligated units dissociate from the target and then serve as
"target sequences" for ligation of excess probe pairs. U.S. Pat.
No. 4,883,750 describes a method similar to LCR for binding probe
pairs to a target sequence.
[0199] Qbeta Replicase, described in PCR Application No.
PCI/US87/00880, may also be used as still another amplification
method in the present invention. In this method, a replicative
sequence of RNA that has a region complementary to that of a target
is added to a sample in the presence of an RNA polymerase. The
polymerase will copy the replicative sequence that can then be
detected.
[0200] An isothermal amplification method, in which restriction
endonucleases and ligases are used to achieve the amplification of
target molecules that contain nucleotide
5'-[alpha-thio]-triphosphates in one strand of a restriction site
may also be useful in the amplification of nucleic acids in the
present invention, Walker et al., (1992).
[0201] Strand Displacement Amplification (SDA) is another method of
carrying out isothermal amplification of nucleic acids which
involves multiple rounds of strand displacement and synthesis,
i.e., nick translation. A similar method, called Repair Chain
Reaction (RCR), involves annealing several probes throughout a
region targeted for amplification, followed by a repair reaction in
which only two of the four bases are present. The other two bases
can be added as biotinylated derivatives for easy detection. A
similar approach is used in SDA. Target specific sequences can also
be detected using a cyclic probe reaction (CPR). In CPR, a probe
having 3' and 5' sequences of non-specific DNA and a middle
sequence of specific RNA is hybridized to DNA that is present in a
sample. Upon hybridization, the reaction is treated with RNase H,
and the products of the probe identified as distinctive products
that are released after digestion. The original template is
annealed to another cycling probe and the reaction is repeated.
[0202] Still another amplification methods described in GB
Application No. 2 202 328, and in PCT Application No.
PCT/US89/01025, each of which is incorporated herein by reference
in its entirety, may be used in accordance with the present
invention. In the former application, "modified" primers are used
in a PCR-like, template- and enzyme-dependent synthesis. The
primers may be modified by labeling with a capture moiety (e.g.,
biotin) and/or a detector moiety (e.g., enzyme). In the latter
application, an excess of labeled probes are added to a sample. In
the presence of the target sequence, the probe binds and is cleaved
catalytically. After cleavage, the target sequence is released
intact to be bound by excess probe. Cleavage of the labeled probe
signals the presence of the target sequence.
[0203] Other nucleic acid amplification procedures include
transcription-based amplification systems (TAS), including nucleic
acid sequence based amplification (NASBA) and 3SR (Kwoh et al.,
1989; Gingeras et al., PCT Application WO 88/10315, incorporated
herein by reference in their entirety). In NASBA, the nucleic acids
can be prepared for amplification by standard phenol/chloroform
extraction, heat denaturation of a clinical sample, treatment with
lysis buffer and minispin columns for isolation of DNA and RNA or
guanidinium chloride extraction of RNA. These amplification
techniques involve annealing a primer which has target specific
sequences. Following polymerization, DNA/RNA hybrids are digested
with RNase H while double stranded DNA molecules are heat denatured
again. In either case the single stranded DNA is made fully double
stranded by addition of second target specific primer, followed by
polymerization. The double-stranded DNA molecules are then multiply
transcribed by an RNA polymerase such as T7 or SP6. In an
isothermal cyclic reaction, the RNA's are reverse transcribed into
single stranded DNA, which is then converted to double stranded
DNA, and then transcribed once again with an RNA polymerase such as
T7 or SP6. The resulting products, whether truncated or complete,
indicate target specific sequences.
[0204] Davey et al., EPO No. 329 822 (incorporated herein by
reference in its entirety) disclose a nucleic acid amplification
process involving cyclically synthesizing single-stranded RNA
("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be
used in accordance with the present invention. The ssRNA is a
template for a first primer oligonucleotide, which is elongated by
reverse transcriptase (RNA-dependent DNA polymerase). The RNA is
then removed from the resulting DNA:RNA duplex by the action of
ribonuclease H(RNase H, an RNase specific for RNA in duplex with
either DNA or RNA). The resultant ssDNA is a template for a second
primer, which also includes the sequences of an RNA polymerase
promoter (exemplified by T7 RNA polymerase) 5' to its homology to
the template. This primer is then extended by DNA polymerase
(exemplified by the large "Klenow" fragment of E. coli DNA
polymerase 1), resulting in a double-stranded DNA ("dsDNA")
molecule, having a sequence identical to that of the original RNA
between the primers and having additionally, at one end, a promoter
sequence. This promoter sequence can be used by the appropriate RNA
polymerase to make many RNA copies of the DNA. These copies can
then re-enter the cycle leading to very swift amplification. With
proper choice of enzymes, this amplification can be done
isothermally without addition of enzymes at each cycle. Because of
the cyclical nature of this process, the starting sequence can be
chosen to be in the form of either DNA or RNA.
[0205] Miller et al., PCT Application WO 89/06700 (incorporated
herein by reference in its entirety) disclose a nucleic acid
sequence amplification scheme based on the hybridization of a
promoter/primer sequence to a target single-stranded DNA ("ssDNA")
followed by transcription of many RNA copies of the sequence. This
scheme is not cyclic, ie., new templates are not produced from the
resultant RNA transcripts. Other amplification methods include
"RACE" and "one-sided PCR" (Frohman, M. A., In: PCR PROTOCOLS: A
GUIDE TO METHODS AND APPLICATIONS, Academic Press, N.Y., 1990;
Ohara et al., 1989; each herein incorporated by reference in their
entirety).
[0206] Methods based on ligation of two (or more) oligonucleotides
in the presence of nucleic acid having the sequence of the
resulting "di-oligonucleotide", thereby amplifying the
di-oligonucleotide, may also be used in the amplification step of
the present invention. Wu et al., (1989), incorporated herein by
reference in its entirety.
[0207] (iii) Southern/Northern Blotting
[0208] Blotting techniques are well known to those of skill in the
art. Southern blotting involves the use of DNA as a target, whereas
Northern blotting involves the use of RNA as a target. Each provide
different types of information, although cDNA blotting is
analogous, in many aspects, to blotting or RNA species.
[0209] Briefly, a probe is used to target a DNA or RNA species that
has been immobilized on a suitable matrix, often a filter of
nitrocellulose. The different species should be spatially separated
to facilitate analysis. This often is accomplished by gel
electrophoresis of nucleic acid species followed by "blotting" on
to the filter.
[0210] Subsequently, the blotted target is incubated with a probe
(usually labeled) under conditions that promote denaturation and
rehybridization. Because the probe is designed to base pair with
the target, the probe will binding a portion of the target sequence
under renaturing conditions. Unbound probe is then removed, and
detection is accomplished as described above.
[0211] (iv) Separation Methods
[0212] It normally is desirable, at one stage or another, to
separate the amplification product from the template and the excess
primer for the purpose of determining whether specific
amplification has occurred. In one embodiment, amplification
products are separated by agarose, agarose-acrylamide or
polyacrylamide gel electrophoresis using standard methods. See
Sambrook et al., 1989.
[0213] Alternatively, chromatographic techniques may be employed to
effect separation. There are many kinds of chromatography which may
be used in the present invention: adsorption, partition,
ion-exchange and molecular sieve, and many specialized techniques
for using them including column, paper, thin-layer and gas
chromatography (Freifelder, 1982).
[0214] (v) Detection Methods
[0215] Products may be visualized in order to confirm amplification
of the marker sequences. One typical visualization method involves
staining of a gel with ethidium bromide and visualization under UV
light. Alternatively, if the amplification products are integrally
labeled with radio- or fluorometrically-labeled nucleotides, the
amplification products can then be exposed to x-ray film or
visualized under the appropriate stimulating spectra, following
separation.
[0216] In one embodiment, visualization is achieved indirectly.
Following separation of amplification products, a labeled nucleic
acid probe is brought into contact with the amplified marker
sequence. The probe preferably is conjugated to a chromophore but
may be radiolabeled. In another embodiment, the probe is conjugated
to a binding partner, such as an antibody or biotin, and the other
member of the binding pair carries a detectable moiety.
[0217] In one embodiment, detection is by a labeled probe. The
techniques involved are well known to those of skill in the art and
can be found in many standard books on molecular protocols. See
Sambrook et al., 1989. For example, chromophore or radiolabel
probes or primers identify the target during or following
amplification.
[0218] One example of the foregoing is described in U.S. Pat. No.
5,279,721, incorporated by reference herein, which discloses an
apparatus and method for the automated electrophoresis and transfer
of nucleic acids. The apparatus permits electrophoresis and
blotting without external manipulation of the gel and is ideally
suited to carrying out methods according to the present
invention.
[0219] In addition, the amplification products described above may
be subjected to sequence analysis to identify specific kinds of
variations using standard sequence analysis techniques. Within
certain methods, exhaustive analysis of genes is carried out by
sequence analysis using primer sets designed for optimal sequencing
(Pignon et al, 1994). The present invention provides methods by
which any or all of these types of analyses may be used. Using the
sequences disclosed herein, oligonucleotide primers may be designed
to permit the amplification of sequences throughout the GA21, GG25,
GJ11 and FI117 transformation events, as well as flanking genomic
regions, which may then be analyzed by direct sequencing.
[0220] (vi) Design and Theoretical Considerations for Relative
Quantitative RT-PCR
[0221] Reverse transcription (RT) of RNA to cDNA followed by
relative quantitative PCR (RT-PCR) can be used to determine the
relative concentrations of specific mRNA species isolated from
plants. By determining that the concentration of a specific mRNA
species varies, it is shown that the gene encoding the specific
mRNA species is differentially expressed.
[0222] In PCR, the number of molecules of the amplified target DNA
increase by a factor approaching two with every cycle of the
reaction until some reagent becomes limiting. Thereafter, the rate
of amplification becomes increasingly diminished until there is no
increase in the amplified target between cycles. If a graph is
plotted in which the cycle number is on the X axis and the log of
the concentration of the amplified target DNA is on the Y axis, a
curved line of characteristic shape is formed by connecting the
plotted points. Beginning with the first cycle, the slope of the
line is positive and constant. This is said to be the linear
portion of the curve. After a reagent becomes limiting, the slope
of the line begins to decrease and eventually becomes zero. At this
point the concentration of the amplified target DNA becomes
asymptotic to some fixed value. This is said to be the plateau
portion of the curve.
[0223] The concentration of the target DNA in the linear portion of
the PCR amplification is directly proportional to the starting
concentration of the target before the reaction began. By
determining the concentration of the amplified products of the
target DNA in PCR reactions that have completed the same number of
cycles and are in their linear ranges, it is possible to determine
the relative concentrations of the specific target sequence in the
original DNA mixture. If the DNA mixtures are cDNAs synthesized
from RNAs isolated from different tissues or cells, the relative
abundances of the specific mRNA from which the target sequence was
derived can be determined for the respective tissues or cells. This
direct proportionality between the concentration of the PCR
products and the relative mRNA abundances is only true in the
linear range of the PCR reaction.
[0224] The final concentration of the target DNA in the plateau
portion of the curve is determined by the availability of reagents
in the reaction mix and is independent of the original
concentration of target DNA. Therefore, the first condition that
must be met before the relative abundances of a mRNA species can be
determined by RT-PCR for a collection of RNA populations is that
the concentrations of the amplified PCR products must be sampled
when the PCR reactions are in the linear portion of their
curves.
[0225] The second condition that must be met for an RT-PCR
experiment to successfully determine the relative abundances of a
particular mRNA species is that relative concentrations of the
amplifiable cDNAs must be normalized to some independent standard.
The goal of an RT-PCR experiment is to determine the abundance of a
particular mRNA species relative to the average abundance of all
mRNA species in the sample.
[0226] Most protocols for competitive PCR utilize internal PCR
standards that are approximately as abundant as the target. These
strategies are effective if the products of the PCR amplifications
are sampled during their linear phases. If the products are sampled
when the reactions are approaching the plateau phase, then the less
abundant product becomes relatively over represented. Comparisons
of relative abundances made for many different RNA samples, such as
is the case when examining RNA samples for differential expression,
become distorted in such a way as to make differences in relative
abundances of RNAs appear less than they actually are. This is not
a significant problem if the internal standard is much more
abundant than the target. If the internal standard is more abundant
than the target, then direct linear comparisons can be made between
RNA samples.
[0227] The above discussion describes theoretical considerations
for an RT-PCR assay for plant tissue. The problems inherent in
plant tissue samples are that they are of variable quantity (making
normalization problematic), and that they are of variable quality
(necessitating the co-amplification of a reliable internal control,
preferably of larger size than the target). Both of these problems
are overcome if the RT-PCR is performed as a relative quantitative
RT-PCR with an internal standard in which the internal standard is
an amplifiable cDNA fragment that is larger than the target cDNA
fragment and in which the abundance of the mRNA encoding the
internal standard is roughly 5-100 fold higher than the mRNA
encoding the target. This assay measures relative abundance, not
absolute abundance of the respective mRNA species.
[0228] Other studies may be performed using a more conventional
relative quantitative RT-PCR assay with an external standard
protocol. These assays sample the PCR products in the linear
portion of their amplification curves. The number of PCR cycles
that are optimal for sampling must be empirically determined for
each target cDNA fragment. In addition, the reverse transcriptase
products of each RNA population isolated from the various tissue
samples must be carefully normalized for equal concentrations of
amplifiable cDNAs. This consideration is very important since the
assay measures absolute mRNA abundance. Absolute mRNA abundance can
be used as a measure of differential gene expression only in
normalized samples. While empirical determination of the linear
range of the amplification curve and normalization of cDNA
preparations are tedious and time consuming processes, the
resulting RT-PCR assays can be superior to those derived from the
relative quantitative RT-PCR assay with an internal standard.
[0229] One reason for this advantage is that without the internal
standard/competitor, all of the reagents can be converted into a
single PCR product in the linear range of the amplification curve,
thus increasing the sensitivity of the assay. Another reason is
that with only one PCR product, display of the product on an
electrophoretic gel or another display method becomes less complex,
has less background and is easier to interpret.
[0230] (vii) Chip Technologies
[0231] Specifically contemplated by the present inventors are
chip-based DNA technologies such as those described by Hacia et al.
(1996) and Shoemaker et al. (1996). Briefly, these techniques
involve quantitative methods for analyzing large numbers of genes
rapidly and accurately. By tagging genes with oligonucleotides or
using fixed probe arrays, one can employ chip technology to
segregate target molecules as high density arrays and screen these
molecules on the basis of hybridization. See also Pease et al.
(1994); Fodor et al. (1991).
[0232] IX. Regeneration of Plants From Transformed Cells
[0233] For use in agriculture, transformation of cells in vitro is
only one step toward commercial utilization of these new methods.
Plants must be regenerated from the transformed cells, and the
regenerated plants must be developed into full plants capable of
growing crops in open fields. For this purpose, fertile corn plants
are required.
[0234] During suspension culture development, small cell aggregates
(10-100 cells) are formed, apparently from larger cell clusters,
giving the culture a dispersed appearance. Upon plating these cells
to solid media, somatic embryo development can be induced, and
these embryos can be matured, germinated and grown into fertile
seed-bearing plants. Alternatively, callus cells growing on solid
culture medium can be induced to form somatic embryos from which
fertile seed bearing plants may develop. The characteristics of
embryogenicity, regenerability, and plant fertility are gradually
lost as a function of time in suspension culture. Cryopreservation
of suspension cells arrests development of the culture and prevents
loss of these characteristics during the cryopreservation
period.
[0235] X. Glyphosate Induced Male-Sterility in GJ11 and GG25
[0236] As demonstrated below, specific applications of glyphosate
may be used to induce male-sterility in corn plants containing one
or more of a particular transformation event, such as, for example,
the GJ11 or GG25 transformation events. A variety of different
parameters of glyphosate application may be used and still induce
male-sterility in plants having a GG25, GJ11 or other similar
transformation event, while at the same time maintaining female
fertility. Treatment will preferably occur at the V4 or later stage
of development, and may occur up to and including any time before
pollen shed (stage VT). Specific times in development which may be
used include, for example, V4, V5, V6, V7, V8, V9, V10, V11, V12,
V13, V14, V15, V16, V17, V18, and any later stage which is prior to
pollen shed. In particular embodiments, the V12-V14, V15-V17 and
V18-VT ranges may be preferred. It also is contemplated that one
may wish to make more than one glyphosate application, for example
glyphosate applications may be made at the V12 and V15 stages.
Application rates used may vary. Useful with the current invention
will be the equivalent of an over-the-top application rate of
between and including 8 ounces per acre and 96 ounces per acre of
glyphosate (e.g. ROUNDUP ULTRA.TM.). Specifically contemplated for
use are all concentrations between about 8 ounces and about 96
ounces per acre including about 8, 12, 16, 20, 24, 28, 32, 36, 40,
44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92 and 96 ounces
per acre. Concentrations deemed particularly useful include, for
example, about 32, 64 and 96 ounces per acre. Alternatively, it is
contemplated that other concentrations of glyphosate may be used
successfully with the current invention; however, such applications
will be less preferred for use with the present invention.
[0237] (i) Utilization of Herbicide Inducible Male-Sterility in
Breeding Programs
[0238] Corn has a diploid phase which means two conditions of a
gene (two alleles) occupy each locus (position on a chromosome). If
the alleles are the same at a locus, there is said to be
homozygosity. If they are different, there is said to be
heterozygosity. In a completely inbred plant, all loci are
homozygous. Because many loci when homozygous are deleterious to
the plant, in particular leading to reduced vigor, less kernels,
weak and/or poor growth, use of inbreds directly by the farmer is
not preferred. Under some conditions, heterozygous advantage at
some loci effectively bars perpetuation of homozygosity. In
general, hybrid maize will demonstrate greater vigor than will
inbreds. Production of hybrids will therefore be of great interest
to the breeder and grower.
[0239] One important application of the inducible male-sterility of
the transformation events of the current invention will be in the
production of hybrid corn seed. For this use, parental plants are
planted in pollinating proximity to each other in alternating rows,
in blocks or in any other convenient planting pattern. One of the
plants, the female parent, will typically comprise a GG25 or GJ11
transformation event or a similar transformation event
demonstrating male-sterility; while the plant used as the male
parent will be glyphosate resistant and will preferably comprise a
GA21, FI117 or similar transformation event conferring male and
female-fertility following glyphosate application. A preferred male
parent will comprise a GA21 transformation event.
[0240] For hybrid production the male and female parents are
typically different elite inbreds derived from different heterotic
backgrounds into which one or more appropriate transformation
events have been backcrossed. Plants of both parental parents are
then cultivated and allowed to grow until the time of flowering.
During this time of cultivation, and prior to pollen shed, one or
more glyphosate applications are made, thereby inducing
male-sterility in plants comprising a GG25, GJ11 or similar
transformation event. Advantageously, during this growth stage,
plants are in general treated with fertilizer and/or other
agricultural chemicals as considered appropriate by the grower.
[0241] Following sterilization, hybridization and fertilization
takes place. Corn plants (Zea mays L) can be crossed by either
natural or mechanical techniques. Natural pollination occurs in
corn when wind blows pollen from the tassels to the silks that
protrude from the tops of the incipient ears. Artificially directed
pollination can be effected either by controlling the types of
pollen that can blow onto the silks or by pollinating by hand. In
conventional plant breeding schemes, at the time of flowering, the
tassels of all the parental plants employed as the female parent
are typically removed. The detasseling can be achieved manually or
by machine, if desired. This technique, while effective, is
extremely labor intensive and greatly increases the overall cost of
hybrid seed production. Alternatively, conventional nuclear or
cytoplasmic or male sterility systems may be used, but such systems
will generally complicate efforts to perpetuate specific inbred
lines.
[0242] In the current invention, the female parent plants will
comprise a GG25 or GJ11 transformation event or another event with
similar properties and are treated with glyphosate at the V5 or
later stage, causing male-sterility in the plants and thereby
avoiding the need for detasseling. This treatment can be carried
out on individual plants, but will more preferably be an
over-the-top treatment of the entire field of male and female
parental plants. In this case, it will be necessary for both male
and female parent plants to be glyphosate resistant and male and
female-fertile, respectively, under the glyphosate application
conditions used to cause male-sterility. An appropriate male parent
will, therefore, be fully fertile under the glyphosate application
conditions which are used to induce male-sterility in the female
parent. Alternatively, the male parent may be excluded from the
glyphosate treatment, and therefore potentially any maize plant
used as the male parent. Exemplary male parents which may be
treated with glyphosate are maize plants having a GA21 or FI117
transformation event, with GA21 being most preferred.
[0243] The plants are allowed to continue to grow and natural
cross-pollination occurs as a result of the action of wind, which
is normal in the pollination of grasses, including corn. As a
result of the induced male-sterility of the female parent plant,
all the pollen from the male parent plant is available for
pollination because tassels, and thereby pollen bearing flowering
parts, have been previously sterilized from all plants being used
as the female in the hybridization. Of course, during this
hybridization procedure, the parental varieties are grown such that
they are isolated from other corn fields to minimize or prevent any
accidental contamination of pollen from foreign sources in
non-glyphosate treated fields. These isolation techniques are well
within the skill of those skilled in this art.
[0244] Both parental inbred plants of corn may be allowed to
continue to grow until maturity or the male rows may be destroyed
after flowering is complete. Only the ears from the female inbred
parental plants are harvested to obtain seeds of a novel F.sub.1 or
other type of hybrid. The novel hybrid seed produced can then be
planted in a subsequent growing season with the desirable
characteristics in terms of hybrid corn plants providing improved
grain yields and the other desirable characteristics disclosed
herein, being achieved. The collected seed, therefore, represents a
valuable commercial product which can be sold to farmers, employed
in further breeding programs, directly planted in the field by the
breeder, or processed.
[0245] In one embodiment, corn seed prepared by such a process is a
first generation seed capable of being grown into an F.sub.1 hybrid
corn plant prepared by a process wherein both the first and second
parent corn plants are inbred corn plants into which the
appropriate transformation events of the current invention have
been backcrossed. In another embodiment, one or both of the first
and second parent corn plants can be hybrids having the appropriate
transformation events.
[0246] Where an inbred corn plant comprising a GG25, GJ11 or other
transformation event with a similar phenotype is crossed with
another, different, corn inbred seed capable of growing into a
first generation (F.sub.1) corn hybrid plant is produced. This
F.sub.1 seed, the F.sub.1 hybrid corn plants grown therefrom, and
seed of that F.sub.1 hybrid corn plant are contemplated as aspects
of the present invention. The goal of a process of producing an
F.sub.1 hybrid is to manipulate the genetic complement of corn to
generate new combinations of genes which interact to yield new or
improved traits (phenotypic characteristics). A process of
producing an F.sub.1 hybrid typically begins with the production of
one or more inbred plants. Those plants are produced by repeated
crossing of ancestrally related corn plants to try and concentrate
certain genes within the inbred plants. Therefore, any inbred
comprising a transformation event of the current invention is also
part of the invention In a preferred embodiment, crossing comprises
the steps of:
[0247] (a) planting in pollinating proximity seeds of a first and a
second parent corn plant, the first parent corn plant preferably
being an inbred comprising a GG25, GJ11 or other transformation
event conferring a similar phenotype, and the second parent
preferably having a FI117, GA21 or other transformation event
conferring a similar phenotype;
[0248] (b) cultivating or growing the seeds of the first and second
parent corn plants;
[0249] (c) applying 8 to 96 ounces per acre of glyphosate (ROUNDUP
ULTRA.TM.) to the parent corn plants between the V8 and VT stages
of development;
[0250] (d) allowing cross-pollination to occur between the first
and second parent corn plant;
[0251] (e) harvesting seeds produced on the first plant; and, where
desired,
[0252] (f) growing the harvested seed into a corn plant.
[0253] The utility of the methods and transformation events of the
current invention also extends to crosses with other species.
Commonly, suitable species will be of the family Graminaceae, and
especially of the genera Zea, Tripsacum, Coix, Schlerachne,
Polytoca, Chionachne, and Trilobachne, of the tribe Maydeae. Of
these, Zea and Tripsacum, are most preferred. Potentially suitable
for crosses with corn plants comprising transformation events of
the current invention can be the various varieties of grain
sorghum, Sorghum bicolor (L.) Moench.
[0254] (ii) Use of Herbicide Applications for Seed Purity
[0255] The current invention may also be used to cause or ensure
genetic purity in breeding protocols. It is specifically
contemplated that, by treating a field with glyphosate, pollen
grains which do not have an allele comprising a GA21, FI117 or
similar transformation event will be sterilized. Thus, through the
appropriate use of glyphosate treatments on specific transgenic
plants, one could greatly enhance the obtained seed purity for the
resistance allele. This could be used to speed the introgression of
a GA21, FI117, or other transformation event which provides pollen
with resistance a particular herbicide, into a particular genetic
background. Through the effective elimination of pollen grains
lacking the herbicide resistance trait, non-resistance alleles will
be eliminated from the cross. The net result is that a plant being
hemizygous for a particular allele can be made to act in a cross as
if a homozygote, with regard to the resistance trait. This can
speed in the introgression of the trait into a particular genetic
line, and can also reduce the time needed in plant breeding, by
eliminating the need for production of herbicide resistance allele
homozygotes to use in hybrid production. Further, through
application of glyphosate to plants grown from the seed produced,
one may also determine the relative proportion, and therefore the
genetic purity, of seed having inherited at least a first herbicide
resistance transformation event.
[0256] In order to use glyphosate to selectively render pollen not
having the desired herbicide resistance transformation event
incapable of fertilizing female reproductive structures, one would
use a protocol similar to that used for inducible male-sterility
aided hybrid production. More specifically, one may apply from 8 to
96 ounces of glyphosate over-the-top to plants which have at least
one copy of the resistance allele. Timing of treatments would be
prior to pollen shed, between the V5 and VT stages of
development.
[0257] Once seed having a herbicide resistance allele is produced,
seed purity may be measured by treating a selected number of plants
grown from the seed with herbicide. Through determinations of the
number of plants which are sensitive or resistant to the herbicide,
one can determine the relative purity of the seed. Potentially, any
herbicide and the corresponding herbicide resistance allele may be
used for this purpose. Specific examples include a mutant EPSPS
gene, a phosphinothricin acetyltransferase gene conferring
glufosinate resistance, a mutant acetolactate synthase gene (ALS)
gene conferring resistance to imidazolinone or sulphonylurea
herbicides, a neo gene which codes for kanamycin and G418
resistance, a nitrilase gene which confers resistance to bromoxynil
and a DHFR gene conferring methotrexate resistance.
[0258] (iii) Applicability of Herbicide Induced Male-Sterility
[0259] It is specifically contemplated by the inventors that the
inducible male-sterility of the current invention may find
applicability to species other than maize and to herbicide
resistance alleles other than EPSPS. More particularly, it is
believed that the glyphosate induceable nature of male-sterility in
plants having the C-G25 and GJ11 transformation events relative to
the lack of male-sterility in GA21 and FI117 plants is a result of
promoter function in expression of the resistance protein, in this
case a mutant EPSPS. It is believed that the rice-actin promoter in
FI117 and GA21 more efficiently drives expression of the mutant
EPSPS gene in pollen than do the maize histone promoter and
CaMV35S-Arabidopsis histone promoter of GG25 and GJ11,
respectively. The result is that pollen from FI117 and GA21
exhibits a tolerance to glyphosate which is substantially enhanced
relative to the pollen of GG25 and GJ11 plants, or plants lacking a
mutant EPSPS allele.
[0260] One may, therefore, through selection of a promoter which is
poorly expressed in pollen, intentionally engineer herbicide
resistant plants in which male-sterility can be induced through
applications of herbicides. One may additionally, by use of the
same resistance gene, but which is operably linked to a
constitutive promoter expressed more efficiently in pollen, also
obtain plants of the same species which have resistance to the same
herbicide but are not inducably male sterile. Species other than
maize for which this technique is deemed to be particularly suited
include sorghum, barley, oat, wheat, rice, and soybean. Herbicide
resistance alleles other than an EPSPS gene which are deemed
particularly suited for this purpose include a phosphinothricin
acetyltransferase gene conferring glufosinate resistance, a mutant
acetolactate synthase gene (ALS) gene conferring resistance to
imidazolinone or sulphonylurea herbicides, a neo gene which codes
for kanamycin and G418 resistance, a nitrilase gene which confers
resistance to bromoxynil and a DHFR gene conferring methotrexate
resistance.
[0261] XI. Definitions
[0262] Female Reproductive Herbicide Tolerance: a plant exhibiting
this trait will remain female fertile following treatment of the
plant with an application of herbicide which is capable of causing
female-sterility in plants not exhibiting the trait.
[0263] Inviable Pollen: pollen which is not capable of fertilizing
a plant to produce seed.
[0264] Male Reproductive Herbicide Tolerance: a characteristic in
which a plant may be treated with an application of herbicide and
remain male-fertile, the herbicide application being capable of
causing male-sterility in non-male reproductively tolerant
plants.
[0265] Male-Sterile: a male-sterile plant is one which is not
capable of self fertilization or fertilization of other plants to
produce seeds.
[0266] Vegetative Herbicide Tolerance: a plant exhibiting this
trait is capable of being treated and not killed by an application
rate of herbicide which is otherwise capable of killing the
corresponding non-vegetatively herbicide tolerant plant.
[0267] XII. Deposit Information
[0268] A deposit of seeds comprising the GJ11, FI117, GG25 and GA21
transformation events has been made with the American Type Culture
Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., 20852. The
date of deposit was May 14, 1997. The ATCC accession numbers for
seed of maize plants comprising the GJ11, FI117, GG25 and GA21
transformation events are: ATCC 209030, ATCC 209031, ATCC 209032,
and ATCC 209033, respectively. All restrictions upon the deposit
have been removed, and the deposit is intended to meet all of the
requirements of 37 C.F.R. .sctn. 1.801-1.809. The deposit will be
maintained in the depository for a period of 30 years, or 5 years
after the last request, or for the effective life of the patent,
whichever is longer, and will be replaced as necessary during that
period.
XIII. EXAMPLES
[0269] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the concept, spirit and scope
of the invention. More specifically, it will be apparent that
certain agents which are both chemically and physiologically
related may be substituted for the agents described herein while
the same or similar results would be achieved. All such similar
substitutes and modifications apparent to those skilled in the art
are deemed to be within the spirit, scope and concept of the
invention as defined by the appended claims.
Example 1
Initiation and Maintenance of Cell Line AT824
[0270] This example describes the initiation and maintenance of
cell line AT824, which has been used routinely for transformation
experiments. Immature embryos (0.5-1.0 mm) were excised from the
B73-derived inbred line AT and cultured on N6 medium with 100 .mu.M
silver nitrate, 3.3 mg/L dicamba, 3% sucrose and 12 mM proline
(2004). Six months after initiation type I callus was transferred
to medium 2008. Two months later type I callus was transferred to a
medium with a lower concentration of sucrose (279). A sector of
type II callus was identified 17 months later and was transferred
to 279 medium. This cell line is uniform in nature, unorganized,
rapid growing, and embryogenic. This culture was desirable in the
context of this invention as it is easily adaptable to culture in
liquid or on solid medium.
[0271] The first suspension cultures of AT824 were initiated 31
months after culture initiation. Suspension cultures may be
initiated in a variety of culture media including media containing
2,4-D as well as dicamba as the auxin source, e.g., media
designated 210, 401, 409, 279. Cultures are maintained by transfer
of approximately 2 ml packed cell volume to 20 ml fresh culture
medium at 3 2 day intervals. AT824 can be routinely transferred
between liquid and solid culture media with no effect on growth or
morphology.
[0272] Suspension cultures of AT824 were initially cryopreserved
33-37 months after culture initiation. The survival rate of this
culture was improved when it was cryopreserved following three
months in suspension culture. AT824 suspension cultures have been
cryopreserved and reinitiated from cryopreservation at regular
intervals since the initial date of freezing. Repeated cycles of
freezing have not affected the growth or transformability of this
culture.
Example 2
Generation of Glyphosate Resistant Line GA21 by Microprojectile
Bombardment of AT824 Cells
[0273] The mutant maize EPSPS gene was introduced into AT824
suspension culture cells via microprojectile bombardment,
essentially as described by U.S. Pat. No. 5,554,798 and U.S. patent
application No. 08/113,561, filed Aug. 25, 1993, which are both
specifically incorporated herein by reference in their entirety. In
this example, the mutant maize EPSPS gene was derived from plasmid
pDPG434 (FIG. 3). Plasmid pDPG434 contains a maize EPSPS gene with
two amino acid changes, Thr to Ile at position 102 and Pro to Ser
at position 106. An approximately 3.4 kb NotI restriction fragment
containing the mutant maize EPSPS expression cassette of pPDG434
was used for transformation. The mutant maize EPSPS expression
cassette contains a rice actin promoter and the nos 3' end.
[0274] Suspension culture AT824 (described in example 1) was
subcultured to fresh medium 409 3 days prior to particle
bombardment. Cells were plated on solid 279 medium 0-8 hours before
bombardment (about 0.5 ml packed cell volume per filter).
[0275] DNA was precipitated onto gold particles as follows. A stock
solution of gold particles was prepared by adding 60 mg of 0.7
.mu.m or 1 .mu.m gold particles to 1000 .mu.l absolute ethanol and
incubating for at least 3 hours at room temperature followed by
storage at -20.degree. C. Twenty to thirty five .mu.l sterile gold
particles were centrifuged in a microcentrifuge for 1 min. The
supernatant was removed and one ml sterile water was added to the
tube, followed by centrifugation at 2000 rpm for 5 minutes.
Microprojectile particles were resuspended in 30 .mu.l of DNA
solution containing about 10-20 .mu.g of the NotI restricted
pDPG434 mutant EPSPS expression cassette. Two hundred twenty
microliters sterile water, 250 .mu.l 2.5 M CaCl.sub.2 and 50 .mu.l
spermidine were added. The mixture was thoroughly mixed and placed
on ice, followed by vortexing at 4 C for 10 minutes and
centrifugation at 500 rpm for 5 minutes. The supernatant was
removed and the pellet resuspended in 600 .mu.l absolute ethanol.
Following centrifugation at 500 rpm for 5 minutes the pellet was
resuspended in 36 .mu.l of absolute ethanol and was allowed to
settle for 4 minutes. Ten .mu.l of the particle preparation were
dispensed on the surface of the flyer disk and the ethanol was
allowed to dry completely. The particles were then accelerated by a
helium blast of approximately 1100 psi using the DuPont Biolistics
PDS1000He particle bombardment device.
[0276] Following bombardment with gold particles coated with the
pDPG434 expression cassette, AT824 cells were cultured on 279
medium (Table 2) for four days. Subsequently, the cells were
returned to liquid 401 medium (Table 2), at a density of about 2 ml
packed cell volume (PCV) per 20 ml, and cultured for four days. The
cells were then transferred, at a density of 2 ml PCV/20 ml, to
fresh 401 medium containing 1 mg/L bialaphos (bialaphos was
accidentally used instead of glyphosate at this stage) and cultured
for four days. The subculture was repeated, this time into 401 plus
1 mM glyphosate, and after four days the cells were plated at a
density of about 0.1 ml PCV per 100.times.20 mm petri dish
containing 279 plus 1 mM glyphosate. Six to eight weeks after
bombardment, glyphosate resistant colonies were removed from the
selection plates and subcultured onto fresh 279 plus 1 mM
glyphosate. Thirty five glyphosate resistant callus lines were
recovered in this example. Approximately 96 plants were regenerated
from 18 of the transgenic callus lines.
Example 3
Stable Transformation of AT824 Cells by Electroporation
[0277] Maize suspension culture cells were enzyme treated and
electroporated using conditions described in Kryzek et al. (U.S.
Pat. No. 5,384,956). AT824 suspension culture cells, three days
post subculture, were sieved through 1000 .mu.m stainless steel
mesh and washed, 1.5 ml packed cells per 10 ml, in incubation
buffer (0.2 M mannitol, 0.1% bovine serum albumin, 80 mM calcium
chloride, and 20 mM 2-(N-morpholino)-ethane sulfonic acid, pH 5.6).
Cells were then treated for 90 minutes in incubation buffer
containing 0.5% pectolyase Y-23 (Seishin Pharmaceutical, Tokyo,
Japan) at a density of 1.5 ml packed cells per 5 ml of enzyme
solution. During the enzyme treatment, cells were incubated in the
dark at approximately 25.degree. C. on a rotary shaker at 60 rpm.
Following pectolyase treatment, cells were washed once with 10 ml
of incubation buffer followed by three washes with electroporation
buffer (10 mM HEPES, 0.4 mM mannitol). Cells were resuspended in
electroporation buffer at a density of 1.5 ml packed cells in a
total volume of 3 ml.
[0278] Linearized plasmid DNA, about 60 .mu.g of NotI excised EPSPS
expression cassette from pDPG427 (GG25) or pDPG443 (GJ11); or 100
.mu.g of whole pDPG165 and pDPG434 (FI117) plasmid DNA (50 .mu.g
from each plasmid), was added to 0.5 ml aliquots of electroporation
buffer. The DNA/electroporation buffer was incubated at room
temperature for approximately 10 minutes. To these aliquots, 0.5 ml
of suspension culture cells/electroporation buffer (containing
approximately 0.25 ml packed cells) were added. Cells and DNA in
electroporation buffer were incubated at room temperature for
approximately 10 minutes. One half ml aliquots of this mixture were
transferred to the electroporation chamber (Puite, 1985) which was
placed in a sterile 60.times.15 mm petri dish. Cells were
electroporated with a 70, 100, or 140 volt (V) pulse discharged
from a 140 microfarad (.mu.f) capacitor.
[0279] Approximately 10 minutes post-electroporation, cells were
diluted with 2.5 ml 409 medium containing 0.3 M mannitol. Cells
were then separated from most of the liquid medium by drawing the
suspension up in a pipet, and expelling the medium with the tip of
the pipet placed against the petri dish to retain the cells. The
cells, and a small amount of medium (approximately 0.2 ml) were
dispensed onto a filter (Whatman #1, 4.25 cm) overlaying solid 279
medium (Table 2) containing 0.3 M mannitol. After about five days,
the tissue and the supporting filters were transferred to 279
medium containing 0.2 M mannitol. After about six days, tissue and
supporting filters were transferred to 279 medium without
mannitol.
Example 4
Regeneration of AT824 Transformants
[0280] Transformants were produced as described in Example 2 and
Example 3. For regeneration, tissue was maintained on maintenance
medium (279) containing 1 mM glyphosate or 1 mg/L bialaphos.
Subsequently transformants were subcultured one to three times, but
usually twice on 189 medium (first passage in the dark and second
passage in low light) and once or twice on 101 medium in petri
dishes before being transferred to 501 or 607 medium in Plant Cons.
Variations in the regeneration protocol are normal based on the
progress of plant regeneration. Hence some of the transformants
were first routinely subcultured on maintenance medium, followed by
twice on 189 medium, once or twice on 101 medium, followed by
transfer to 501 or 607 medium in Plant Cons. As shoots developed on
101 medium, the light intensity was increased by slowly adjusting
the distance of the plates from the light source located overhead.
All subculture intervals were for about 2 weeks at about 24.degree.
C. Transformed plants that developed shoots roots were transferred
to soil.
[0281] Plantlets in soil were incubated in an illuminated growth
chamber and conditions were slowly adjusted to adapt or condition
the plantlets to the drier and more illuminated conditions of the
greenhouse. After adaptation/conditioning in the growth chamber,
plants were transplanted individually to 5 gallon pots of soil in
the greenhouse.
Example 5
Regeneration of Glyphosate Resistant Line FI117 Using Bialaphos
Selection
[0282] Cells of AT824 were electroporated with plasmids pDPG165 and
pDPG434 as described in example 3. In this case, co-transformation
with the bar gene-containing plasmid pDPG165 allowed for selection
on bialaphos. Following recovery and after the tissue had grown for
about four days on 279 medium, the tissue on each filter was
transferred to a flask containing about 20 ml of liquid 401 medium
containing 1 mg/L bialaphos. Four days later, tissue in each flask
was transferred to a new flask containing about 20 ml fresh 401
medium containing 1 mg/L bialaphos. Three days later the cells were
plated at a density of about 0.1 ml PCV per 100.times.20 mm petri
dish containing 279 medium plus 1 mg/L bialaphos. Approximately 34
bialaphos resistant callus lines were selected in this example, at
a frequency of 17 callus lines per electroporation. Approximately
48 plants were regenerated from 18 callus lines. Screening of
plants for glyphosate resistance was subsequently carried out as
described in example 5.
Example 6
Screening Transgenic Plants for Glyphosate Resistance
[0283] Plants regenerated from callus lines GA21, GG25, GJ11, and
FI117 (R.sub.0 generation), which each contained the mutant EPSPS
gene, were crossed to nontransgenic inbred plants in the
greenhouse. The progeny of these crosses were expected to segregate
1:1 for the herbicide resistance trait. Glyphosate resistance was
evaluated in the progeny of the R.sub.0 crosses (R.sub.1
generation) in a greenhouse by application of Roundup.TM. brand
(Monsanto) glyphosate at a rate of 16 oz./acre. Transgenic lines
that exhibited resistance to glyphosate were selected and again
backcrossed to a nontransgenic inbred. The resulting progeny were
then screened for glyphosate resistance in field tests. From these
tests, the GA21, FI117, GG25 and GJ11 transformation events were
selected for further study based their glyphosate resistant
phenotype.
Example 7
Isolation of Genomic Corn DNA
[0284] Glyphosate resistant corn lines GA21, FI117, GG25 and GJ11
were crossed to various inbred lines to facilitate hybrid
development as described in example 14. Genomic DNA used for
Southern blot analyses was isolated from the resulting backcrossed
plants. The backcross populations consisted of plants that were
segregating 1:1 for the GA21, FI117, GG25 or GJ11 insertion.
Positive and negative GA21 segregants were identified by polymerase
chain reaction (PCR) using oligonucleotide primers specific to the
pDPG434 fragment used for transformation. Negative segregants
served as nontransgenic control plants. The PCR primers used for
the analysis spanned the mutant EPSPS-nos junction and generated a
192 bp fragment. The sequence of the upper primer located on the
mutant EPSPS gene is 5'-ACGTACGACGACCACAGGATG-3'. The sequence of
the lower primer located in nos is 5'-GCAAGACCGGCAACAGGATTC-3'.
Genomic DNA was isolated from positive and negative plants as
described in Dellaporta et al., (1983). DNA was isolated from
field-grown and greenhouse-grown plants.
Example 8
DNA Probe Preparation and Hybridization
[0285] DNA fragments used for probe preparation were isolated by
gel-purification of restriction digests of plasmid DNA or were
generated by PCR. The mutant EPSPS PCR fragment used as a probe was
generated using primers that produce a 324 bp fragment internal to
the EPSPS gene. This fragment initiates approximately 400 bp down
stream from the start codon. The primer sequences used to generate
this fragment are: 5'-TTTGGCTCTTGOGGATGTG-3' (upper) and
5'-TTACGCTAGTCTCGGTCCAT-3' (lower). Probes were labeled with
.sup.32P using the random priming method (Boehringer Mannheim) and
purified using Quik-Sep.RTM. spin columns (Isolab Inc., Akron,
Ohio). Blots were prehybridized at 65.degree. C. for 1-2 hours and
hybridized with denatured probe for approximately 18 hours at
65.degree. C. Prehybridization and hybridization solution consisted
of 5.times.SCP, 2.times. Denhardt's Solution, 0.05 M Tris, pH 8.0,
0.2% SDS, 10 mM EDTA, 100 mg/l dextran sulfate, and 125 .mu.g/ml
denatured salmon sperm DNA. Following hybridization, blots were
washed 4 times for 10 min. in 0.25.times.SCP/0.2% SDS. Membranes
were blotted dry and visualized by autoradiography. To reprobe
blots, probes were removed by treating blots in 0.05 M NaOH/0.2%
SDS for 10 min. followed by neutralization in 0.2 M Tris, pH
7.5/0.2% SDS/0.1.times.SCP for 20 minutes at approximately
25.degree. C.
[0286] Approximately 10 .mu.g of genomic DNA was used for each
restriction digest. DNA was digested with restriction enzymes
according to the manufacturer's recommendations (Boehringer
Mannheim, Indianapolis, Ind.). DNA was separated on TAE gels (0.04
M Tris-acetate, 0.001 M EDTA) containing 0.8% agarose. Southern
blotting (Southern, 1975) was performed using MagnachargeTM
membrane (Micron Separations Inc., Westborough, Mass.) and the DNA
was cross-linked to the membrane using UV light and membranes were
baked for 2 hrs. in a vacuum oven at 80.degree. C.
Example 9
Copy Number and Integrity of the Mutant EPSPS Transgene in GA21
[0287] Corn line GA21 was analyzed to determine the number of
insertions of the pDPG434 NotI EPSPS fragment used for
transformation. GA21 genomic DNA was digested with a restriction
enzyme that does not cut within the NotI EPSPS fragment used for
transformation and probed with the entire NotI EPSPS fragment. For
this analysis, GA21 DNA and nontransformed control DNAs were
digested with EcoRV and probed with the NotI EPSPS fragment from
pDPG434. NotI digested pDPG434 was included as a positive control
at the level of approximately one copy per genome. For GA21, a
single band of approximately 15 kb hybridized to the probe,
indicating that a single insertion of the plasmid DNA fragment used
for transformation had occurred (FIG. 5A). Some additional
hybridization was observed in GA21 and nontransformed control DNA;
this result was expected given that the probe used contained the
transit peptide sequence (which includes maize DNA) and the mutant
maize EPSPS gene. Both of these sequences are expected to hybridize
to nontransformed maize DNA due to the presence of endogenous
sequences with homology to the probe sequence.
[0288] To further clarify the presence of a single insertion of the
pDPG434 plasmid fragment in GA21, the probe was removed from the
blot shown in FIG. 5A and the blot was rehybridized using a small
DNA fragment internal to the mutant EPSPS gene. The 324 bp EPSPS
probe hybridized strongly to the same approximately 15 kb band in
GA21 DNA, indicating the presence of a single insertion of the NotI
EPSPS fragment used for transformation (FIG. 5B). Using the 324 bp
EPSPS probe, hybridization to two smaller molecular weight bands
was observed in both GA21 and nontransformed control DNA,
indicating the presence of endogenous copies of the native EPSPS
gene.
[0289] To determine if the mutant EPSPS gene in glyphosate
resistant corn line GA21 was intact and to estimate copy number,
genomic DNA from a GA21 transformant, nontransformed control DNA,
and pDPG434 were digested with EcoRI/XbaI and probed with the 324
bp EPSPS probe. This restriction enzyme digest releases a fragment
of approximately 1.8 kb from pDPG434 that contains the OTP sequence
and the mutant EPSPS gene (FIG. 3). EcoRI/XbaI digested pDPG434 was
run on the gel to approximate one copy of the EcoRI/XbaI OTP-EPSPS
sequence per genome. The 324 bp mutant EPSPS probe was found to
hybridize to an approximately 1.8 kb fragment in GA21 and the
pDPG434 digests, but not in the digest of nontransformed control
DNA. (FIG. 6). This result demonstrates that the 1.8 kb OTP-EPSPS
fragment present on pDPG434 is intact in glyphosate resistant corn
line GA21. Comparison of the hybridization intensity of the pDPG434
digest to the digest of GA21 DNA indicates the presence of
approximately two copies of the OTP-EPSPS sequence in GA21 (FIG.
6).
Example 10
Lack of Plasmid Backbone Sequences in GA21
[0290] To confirm the lack of plasmid backbone sequences containing
the ColE1 origin of replication and the bla gene encoding
.beta.-lactamase, DNA from a transgenic corn line containing a
single copy of bla, DNA from a GA21 plant, and plasmid DNA were
digested with BglII and probed with a 1.7 kb SspI/AflIII fragment
from pBluescript SK(-) (Stratagene, La Jolla, Calif.) containing
ColE1 and bla. The plasmid used, pDPG427, is identical to pDPG434
but contains a maize histone promoter instead of the rice actin
promoter. As expected, no hybridization to the GA21 DNA was
observed. Also as expected, hybridization to an approximately 5 kb
band in the DNA from the bla-positive plant and from pDPG427 was
observed (FIG. 7).
Example 11
Construction of Plasmids pDPG 165, pDPG434 and pDPG443
[0291] DNA segments encoding the bar gene were constructed into
plasmid pDPG165 (FIG. 1) essentially as described in U.S. patent
application Ser. No. 08/113,561, filed Aug. 25, 1993, which is
specifically incorporated herein by reference in its entirety. The
bar gene was cloned from Streptomyces hygroscopicus (White et al.,
1990) and exists as a 559-bp SmaI fragment in the plasmid p114101.
The sequence of the coding region of this gene is identical to that
published (Thompson et al., 1987). To create plasmid pDPG165, the
SmaI fragment from pIJ4104 was ligated into a pUC19-based vector
containing the Cauliflower Mosaic Virus (CaMV) 35S promoter
(derived from pBI221.1. provided by R. Jefferson, Plant Breeding
Institute, Cambridge, England), a polylinker, and the transcript 7
(Tr7) 3' end from Agrobacterium tumefaciens (3' end provided by D.
Stalker, Calgene, Inc., Davis, Calif.).
[0292] The plasmids pDPG434 (FIG. 3) and pDPG443 (FIG. 4) were
constructed by cloning the respective promoters into
SmaI-linearized pDPG425 (FIG. 14). Linearized vectors were treated
with calf alkaline phosphatase to prevent recircularization prior
to ligation. The rice actin promoter and intron were isolated as a
1.5 kb HindIII fragment from pDPG421 (pDM302; Cao et al., Plant
Cell Rep (1992) 11:586-591). The 2.times.35S/Arabidopsis histone
promoter was isolated as a 1.8 kb EcoRI/HindIII fragment from
pDPG405 (FIG. 15). The above mentioned promoter fragments were
T.sub.4 DNA polymerase-treated to create blunt ends prior to
ligation into SmaI linearized pDPG425 (Rhone Poulenc Agrochimie).
The fourth plasmid used, pDPG427 (FIG. 2), was obtained from Rhone
Poulenc Agrochimie. A list of plasmids used in the current
invention as well as the components of the plasmids is given in
Table 4. A list of components of pDPG434 is shown in Table 5.
Example 12
Effect of Glyphosate Application on the Growth and Fertility of
DK580 and DK626 Hybrids of FI117, GA21, GG25 and GJ11
[0293] BC.sub.4 hybrids of DK580 and DK626 were produced containing
one of the FI 17, GA21, GG25 or GJ11 transformation events as
described in example 14. Comparisons of the effect of glyphosate
application on growth (mean extended leaf height) and male
fertility was compared at both the V4 and V8 developmental stage.
The developmental scale that was used to rate the corn plants is
well known in the art, and is described in Special Report No. 48,
Iowa State University of Science and Technology, Cooperative
Extension Service, Ames, Iowa. Each of the hybrids was studied at
both the V4 and V8 stage using 0.times. glyphosate (i.e. water
only), 1.times. glyphosate, or 4.times. glyphosate (the 1.times.
level corresponds to 16 ounces/acre of ROUNDUP ULTRA.TM.).
[0294] Tests were designed as four row, 3 rep., split-split-plot
with main plots as hybrids, subplots as transformant sources (i.e.
GA21, GG25, FI117, and GJ11) and subplots as timing/rate
combinations (FIG. 12). Statistical methods for design and analysis
of data derived from experimental field plots are described in
Gomez and Gomez, (1984). Tests were conducted in Dekalb, Ill., and
Thomasboro, Ill. during 1996. All rows were planted at double
normal planting density, i.e., 60 seeds per row, because hybrids
segregated 1:1 for the glyphosate resistance trait. Sprayed plants
were thinned to 30 plants per row no sooner than 7 days after
application of Roundup at a time when Roundup susceptible plants
could be identified. Unsprayed plots were thinned to 30 plants per
row at the same time. At 5-10 days after herbicide application, the
following data was collected in each row: number of dead plants,
number of damaged plants, and number of normal plants. After
thinning, the mean extended leaf height was measured on 10
resistant plants per plot. During the remainder of the growing
season the following agronomic data was collected: early stand
count, seedling vigor, final stand count, plant height, ear height,
intactness, stay green, number of barren plants, number of
male-sterile plants, number of dropped ears, number of root lodged
plants, number of stalks lodged plants, shelled grain weight,
percent grain moisture at harvest, and test weight.
[0295] The results show that all 4 transformation events gave
significant resistance to glyphosate at both the 1.times. and
4.times. application levels (FIGS. 7A, 7B). Overall, the GA21
transformation event yielded the most efficacious resistance, in
that at the 4.times. application level, 3 of the 4 GA21 treatments
(FIGS. 7A, 7B) had the greatest mean extended leaf heights.
Additionally, all 4 of the GA21 treatments yielded male-fertile
plants. At the V8 stage of application, only GJ11 and GJ25
treatments yielded male sterile plants (FIG. 8B), while at the V4
stage of application all plants were male-fertile (FIG. 8A).
Example 13
Yield Effect of Glyphosate Application on DK580 and DK626 Hybrids
of FI117, GA21, GG25 and GJ11 Transformation Events
[0296] Four DK580 and four DK626 hybrids, each containing a
different mutant EPSPS transgene from one of the GA21, FI117, GJ11
or GG25 transformation events, were field tested for possible
effects on yield with glyphosate application as described in
Example 12, with treatments as shown in FIG. 12. Hybrids were
produced as described in example 14. Yield estimates were computed
using shelled grain weights, adjusted to 15.5% moisture. Data were
analyzed using the SAS PROC MIXED and PROC SUM procedures. Only
hybrids to which no glyphosate was applied were compared in order
to remove any effects of herbicide application rates and/or weed
competition on grain yield. The discussion herein will concentrate
on results relating to grain yield.
[0297] FIG. 9A shows that when glyphosate is applied at the V4
stage, significant decreases in yield are not observed for 3 of the
4 DK580 hybrids. Further, in the case of the GA21 transformation
event, the treatment group had a higher yield, although the
difference was not found to be statistically significant. The
differences in yield between DK580 hybrids with the introgressed
mutant EPSPS transformation event relative to the hybrid without
the event was significant only for the FI117 event. In this case,
the glyphosate resistant hybrid had a higher yield than the
corresponding non-resistant hybrid. The results demonstrate that
glyphosate may be applied to three of the four DK580 hybrids at the
V4 developmental stage without a corresponding decrease in
yield.
[0298] Comparisons of the effect of glyphosate application on yield
in each of the DK626 hybrids at the V8 developmental stage are
given in FIG. 9B. The results demonstrate that even at the V8
stage, no significant loss in yield is observed upon a 4.times.
rate of glyphosate application in either the GA21 or the FI117
introgressed DK626 hybrid. Further, the GA21 hybrid again realized
a gain in yield relative to untreated controls of the same genetic
background.
Example 14
Introgression of GA21, FI117, GG25, and GJ11 Into Elite Inbreds and
Hybrids of Maize
[0299] Backcrossing can be used to improve an inbred plant.
Backcrossing transfers a specific desirable trait from one inbred
source to an inbred that lacks that trait. This can be
accomplished, for example, by first crossing a superior inbred (A)
(recurrent parent) to a donor inbred (non-recurrent parent), which
carries the appropriate gene(s) for the trait in question. The
progeny of this cross are first selected in the resultant progeny
for the desired trait to be transferred from the non-recurrent
parent, then the selected progeny are mated back to the superior
recurrent parent (A). After five or more backcross generations with
selection for the desired trait, the progeny are hemizygous for
loci controlling the characteristic being transferred, in this case
a mutant EPSPS transgene, but are like the superior parent for most
or almost all other genes. The last backcross generation would be
selfed to give progeny which are pure breeding for the gene(s)
being transferred, i.e. a GA21, GJ25, GJ11, and/or FI117
transformation event.
[0300] Therefore, through a series a breeding manipulations, a
selected gene encoding a mutant EPSPS may be moved from one corn
line into an entirely different corn line without the need for
further recombinant manipulation. Introduced transgenes are
valuable in that they behave genetically as any other corn gene and
can be manipulated by breeding techniques in a manner identical to
any other corn gene. Exemplary procedures of this nature have been
successfully carried out by the inventors. In these backcrossing
studies, the transformants GA21, FI117, GG25, and GJ11 were each
introgressed into the elite inbred lines FBLL (U.S. patent
application Ser. No. 08/181,708, filed Jan. 14, 1994) and NL054B
(U.S. patent application Ser. No. 08/595,549, filed Feb. 6, 1996)
by backcrossing, although conversion to many more inbreds is
currently in progress. Using these inbreds as female parents, two
such exemplary hybrids were produced, DK626 and DK580. These
hybrids were field tested for yield and other agronomic
characteristics as well as herbicide tolerance.
[0301] The elite inbreds FBLL and NL054B were each backcrossed four
times to the GA21, FI117, GJ11 and GG25 transformants. At each
backcross generation plants containing the mutant EPSPS gene were
identified based on resistance to a 1.times. application of
glyphosate. Following four generations of backcrossing to a
recurrent elite inbred parent, it is anticipated that the
transformed line will be present in a genetic background that is at
least 93% identical to the recurrent parent (FBLL or NL054B).
Following backcross conversion, the plants were self-pollinated
twice in order to identify plants homozygous for the introgressed
gene of interest, i.e., the GA21, FI117, GJ11 and GG25 insertion
events. Hybrids were produced by crossing the FBLL and NL054B
inbred parents, which contained an insertion event of the GA21,
FI117, GJ11 or GG25, to a non-transformed inbred male parent. DK580
hybrids were produced by a cross of FBLL to MBZA (U.S. patent
application Ser. No. 08/182,616, filed Jan. 14, 1994) and DK626
hybrids were produced by a cross of NL054B by MM402A (U.S. patent
application Ser. No. 08/181,019, filed Jan. 13, 1994), thereby
yielding hybrids which were hemizygous for the respective
transformation event.
Example 15
Marker Assisted Breeding
[0302] The identification of maize lines that are bred for
increased glyphosate resistance may be readily assisted by using a
mutant EPSPS gene integration event from the GA21, GG25, Fi 17 or
GJ11 transformation events. Techniques for isolating nucleic acids
and proteins are well known to those of skill in the art (Sambrook
et al., 1989), and may be used in conjunction with the integration
events of the present invention to selectively segregate plants
that have increased glyphosate resistance.
[0303] It is contemplated that mutant EPSPS gene integration events
will be useful as DNA probes for marker assisted breeding. In the
process of marker assisted breeding DNA sequences are used to
follow desirable agronomic traits (Tanksley et al., 1989) in the
process of plant breeding. Therefore, assays which indicate the
presence mutant EPSPS integration events of the current invention
can be used for identification of plants with enhanced glyphosate
resistance.
[0304] Marker assisted breeding using a mutant EPSPS gene
integration event is undertaken as follows. Seed of plants with
desired yield are planted in soil in the greenhouse or in the
field. Leaf tissue is harvested from the plant for preparation of
DNA at any point in growth at which approximately one gram of leaf
tissue can be removed from the plant without compromising the
viability of the plant. Genomic DNA is isolated using procedure
modified from Shure et al. (1983). Approximately one gram of leaf
tissue from a seedling is lypholyzed overnight in 15 ml
polypropylene tubes. FreezeAried tissue is ground to a power in the
tube using a glass rod. Powdered tissue is mixed thoroughly with 3
ml extraction buffer (7.0 urea, 0.35 M NaCL 0.05 M Tris-HCl ph 8.0,
0.01 M EDTA, 1% sarcosine). Tissue/buffer homogenate is extracted
with 3 ml phenol/chloroform. The aqueous phase is separated by
centrifugation, and precipitated twice using {fraction (1/10)}
volume of 4.4 M ammonium acetate pH 5.2, and an equal volume of
isopropanol. The precipitate is washed with 75% ethanol and
resuspended in 100-500 .mu.l TE (0.01 M Tris-HCl, 0.001 M EDTA, pH
8.0). Genomic DNA is digested with a 3-fold excess of restriction
enzymes, electrophoresed through 0.8% agarose (FMC), and
transferred (Southern, 1975) to Nytran (Schleicher and Schuell)
using 10.times.SCP (20 SCP: 2M NaCl, 0.6 M disodium phosphate, 0.02
M disodium EDTA).
[0305] One of skill in the art will recognize that many different
restriction enzymes will be useful and the choice of restriction
enzyme will depend on the DNA sequence of the mutant EPSPS gene
integration event that is used as a probe and the DNA sequences in
the maize genome surrounding the mutant EPSPS gene integration
event. For a probe, one will want to use DNA or RNA sequences which
will hybridize to DNA from the plasmid DNA of the integration
event. The transformation event-plasmid combinations used herein
are, for example, GA21-pDPG434, GG25-pDPG427, GJ11-pDPG443, and
FI117-pDPG434 and pDPG165. One will select a restriction enzyme
that produces a DNA fragment following hybridization that is
identifiable as that mutant EPSPS gene integration event.
[0306] It is expected that one or more restriction enzymes will be
used to digest genomic DNA either singly or in combinations.
Filters are prehybridized in 6.times.SCP, 10% dextran sulfate, 2%
sarcosine, and 500 .mu.g/ml denatured salmon sperm DNA and
.sup.32P-labeled probe generated by random priming (Feinberg &
Vogelstein, 1983). Hybridized filters are washed in 2.times.SCP, 1%
SDS at 65.degree. for 30 minutes and visualized by autoradiography
using Kodak XAR5 film. Those of skill in the art will recognize
that there are many different ways to isolate DNA from plant
tissues and that there are many different protocols for Southern
hybridization that will produce identical results. Those of skill
in the art will recognize that a Southern blot can be stripped of
radioactive probe following autoradiography and re-probed with a
different mutant EPSPS gene integration event probe. In this manner
one may identify each of the various mutant EPSPS gene integration
events that is present in the plant.
[0307] Each lane of the Southern blot represents DNA isolated from
one plant. Through the use of multiplicity of mutant EPSPS gene
integration events as probes on the same genomic DNA blot, the
integration event composition of each plant may be determined.
Correlations are established between the contributions of
particular integration events to increasing the herbicide
resistance of the plant. Only those plants that contain the desired
combination of integration events are advanced to maturity and used
for pollination. DNA probes corresponding to mutant EPSPS gene
integration events are useful markers during the course of plant
breeding to identify and combine particular integration events
without having to grow the plants and assay the plants for
agronomic performance.
Example 16
General Methods for Assays
[0308] DNA analysis was performed as follows. Genomic DNA was
isolated using a procedure modified from Shure, et al., 1983.
Approximately 1 gm callus tissue was ground to a fine powder in
liquid nitrogen using a mortar and pestle. Powdered tissue was
mixed thoroughly with 4 ml extraction buffer (7.0 M urea, 0.35 M
NaCl, 0.05 M Tris-HCl pH 8.0, 0.01 M EDTA, 1% sarcosine).
Tissue/buffer homogenate was extracted with 4 ml phenol/chloroform.
The aqueous phase was separated by centrifugation, passed through
Miracloth, and precipitated twice using {fraction (1/10)} volume of
4.4 M ammonium acetate, pH 5.2- and an equal volume of isopropanol.
The precipitate was washed with 70% ethanol and resuspended in
200-500:1 TE (0.01 M Tris-HCl, 0.001 M EDTA, pH 8.0). Plant tissue
may also be employed for the isolation of DNA using the foregoing
procedure.
[0309] The presence of a gene in a transformed cell may be detected
through the use of polymerase chain reaction (PCR). Using this
technique specific fragments of DNA can be amplified and detected
following agarose gel electrophoresis. For example the mutant EPSPS
gene may be detected using PCR. Two hundred to 1000 ng genomic DNA
is added to a reaction mix containing 10 mM Tris-HCl pH 8.3, 1.5 mM
MgCl.sub.2, 50 mM KCl, 0.1 mg/ml gelatin, 200 .mu.M each dATP,
dCTP, dGTP, dTTP, 0.5 .mu.M each forward and reverse DNA primers,
20% glycerol, and 2.5 units Taq DNA polymerase. The primer
sequences are (upper) 5'-TTGGCTCTTGGGGATGTG-3' and (lower)
5'-TTACGCTAGTCTCGGTCCAT-3'. The reaction is run in a thermal
cycling machine as follows: 3 minutes at 94 C, 39 repeats of the
cycle 1 minute at 94 C, 1 minute at 50 C, 30 seconds at 72 C,
followed by 5 minutes at 72 C. Twenty .mu.l of each reaction mix is
run on a 3.5% NuSieve gel in TBE buffer (90 mM Tris-borate, 2 mM
EDTA) at 50V for two to four hours. Using these primers a 324 base
pair fragment of the mutant EPSPS transgene is amplified.
[0310] For Southern blot analysis genomic DNA was digested with a
3-fold excess of restriction enzymes, electrophoresed through 0.8%
agarose (FMC), and transferred (Southern, 1975) to Nytran
(Schleicher and Schuell) using 10.times.SCP (20.times.SCP: 2 M
NaCl, 0.6 M disodium phosphate, 0.02 M disodium EDTA). Filters were
prehybridized at 65.degree. C. in 6.times.SCP, 10% dextran sulfate,
2% sarcosine, and 500 .mu.g/ml heparin (Chomet et al., 1987) for 15
min. Filters were hybridized overnight at 65 C in 6.times.SCP
containing 100 .mu.g/ml denatured salmon sperm DNA and
.sup.32P-labeled probe. Filters were washed in 2.times.SCP, 1% SDS
at 65 C for 30 min. and visualized by autoradiography using Kodak
XAR5 film. For rehybridization, the filters were boiled for 10 min.
in distilled H.sub.2O to remove the first probe and then
prehybridized as described above.
Example 17
Weed Control in Agricultural Fields of Glyphosate Resistant Maize
Plants
[0311] Roundup.TM. is a commercial formulation of glyphosate
manufactured and sold by the Monsanto Company. The amount of
Roundup.TM. (glyphosate) which is applied to an agricultural field
in which glyphosate resistant maize plants grow depends on the
particular weed or spectrum of weeds present in the field and for
which control is desired. Herbicide application rates may typically
range from four ounces of Roundup.TM. to 256 ounces Roundup.TM. per
acre (the 1.times. rate is equivalent to 16 ounces per acre of
Roundup.TM., ie., 64 ounces/acre is 4.times.). Preferably, from 8
ounces to 128 ounces per acre of Roundup.TM. are applied to an
agricultural field in which glyphosate resistant maize plants are
present. More preferably, from about 16 ounces to about 64 ounces
per acre of Roundup.TM. may be applied to the field. An application
of Roundup.TM. in excess of the 1.times. rate, including 1.times.,
2.times., 3.times., 4.times. and greater, is sufficient to kill
maize plants which do not have an expressed copy of the mutant
EPSPS gene, and will additionally kill a wide spectrum of
weeds.
[0312] An initial field application of glyphosate is typically
carried out between about the V3 to V5 stages of development and
will typically consist of about a 2.times. application. The
application rate may be increased or decreased as needed, based on
the abundance and/or type of weeds being treated. Depending on the
location of the field and weather conditions, which will influence
weed growth and the type of weed infestation, it may be desirable
to conduct further glyphosate treatments. The second glyphosate
application will typically consist of about a 2.times. glyphosate
application made between the V6 and V8 stage of maturity. Again the
treatment rate may be adjusted based on field conditions. Such
methods of application of herbicides to agricultural crops are well
known in the art and are summarized in general in Anderson,
(1983).
[0313] A farmer may also apply a combination of herbicides
including Roundup.TM., to a field in which glyphosate resistant
maize plants are present. Combination of herbicides are referred to
as "tank mixes." A second herbicide is supplied in combination with
Roundups in order to complement the activity of Roundup.TM. and
thereby increase the efficiency of weed control. For example,
Roundup.TM. may be applied to a field of glyphosate resistant maize
plants in conjunction with a herbicide with residual activity, such
as a triazine herbicide, in order to provide longer lasting weed
control. One herbicide which may be particularly useful in mixture
with glyphosate is acetochlor. It is contemplated that Roundup.TM.
may be applied to an agricultural field comprising glyphosate
resistant maize plants in conjunction with one or more of the
herbicides listed in Table 1. It is understood that the list of
herbicides in Table 1 is not limiting and one of skill in the art
will know the identity of other herbicidal chemicals which a farmer
could apply to a field in combination with Roundup.TM..
[0314] A farmer may wish to apply glyphosate to a field for weed
control at any time during the growth of the corn plant at which
time the farmer desires to control weed growth. Preferably,
glyphosate is applied to the field during vegetative growth of the
maize plants, i.e., prior to the onset of flowering. Roundup.TM.
may be applied to glyphosate resistant plants in the field at any
stage of development, including between the V1 and V10 stages (the
developmental scale is described in, "How a Corn Plant Grows",
Special Report No. 48, Iowa State University of Science and
Technology, Cooperative Extension Service, Ames, Iowa) of
vegetative growth. More preferably, Roundup.TM. is applied to the
field at the V2, V3, V4, V5, V6, V7 or V8 stages of vegetative
growth, and most preferably at the V4, V5, V6, V7 or V8 stages of
growth of the maize plant. Further, multiple applications of
Roundup.TM. may be desired in order to control weed growth. For
example, Roundup.TM. may be applied to the field at both the V4
stage of growth of the glyphosate resistant maize plant and at the
V8 stage of growth. Furthermore, Roundup.TM. may be applied on an
as needed basis in order to control growth of particular weeds when
required.
Example 18
Utilization of Transgenic Crops
[0315] The ultimate goal in plant transformation is to produce
plants which are useful to man. In this respect, transgenic plants
created in accordance with the current invention may be used for
virtually any purpose deemed of value to the grower or to the
consumer. For example, one may wish to harvest seed from transgenic
plants. This seed may in turn be used for a wide variety of
purposes. The seed may be sold to farmers for planting in the field
or may be directly used as food, either for animals or humans.
Alternatively, products may be made from the seed itself Examples
of products which may be made from the seed include, oil, starch,
animal or human food, pharmaceuticals, and various industrial
products. Such products may be made from particular plant parts or
from the entire plant. One product made from the entire plant,
which is deemed of particular value, is silage for animal feed.
[0316] Means for preparing products from plants, such as those that
may be made with the current invention, have been well known since
the dawn of agriculture and will be obvious to those of skill in
the art. Specific methods for crop utilization may be found in, for
example, Sprague and Dudley (1988), and Watson and Ramnstad
(1987).
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Sequence CWU 1
1
5 1 21 DNA Zea mays 1 acgtacgacg accacaggat g 21 2 21 DNA Zea mays
2 gcaagaccgg caacaggatt c 21 3 19 DNA Zea mays 3 tttggctctt
ggggatgtg 19 4 20 DNA Zea mays 4 ttacgctagt ctcggtccat 20 5 570 PRT
Zea mays 5 Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr
Ala Pro 1 5 10 15 Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu
Lys Ser Asn Ala 20 25 30 Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp
Phe Ser Thr Leu Pro Ser 35 40 45 Asn Gly Gly Gly Arg Val Gln Cys
Met Gln Val Trp Pro Ala Tyr Gly 50 55 60 Asn Lys Lys Phe Glu Thr
Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala 65 70 75 80 Pro Thr Val Met
Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gln 85 90 95 Gly Leu
Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg 100 105 110
Ser Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Met Ala Gly 115
120 125 Ala Glu Glu Ile Val Leu Gln Pro Ile Lys Glu Ile Ser Gly Thr
Val 130 135 140 Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile Leu
Leu Ile Ala 145 150 155 160 Ala Leu Ser Glu Gly Thr Thr Val Val Asp
Asn Leu Leu Asn Ser Glu 165 170 175 Asp Val His Tyr Met Leu Gly Ala
Leu Arg Thr Leu Gly Leu Ser Val 180 185 190 Glu Ala Asp Lys Ala Ala
Lys Arg Ala Val Val Val Gly Cys Gly Gly 195 200 205 Lys Phe Pro Val
Glu Asp Ala Lys Glu Glu Val Gln Leu Phe Leu Gly 210 215 220 Asn Ala
Gly Ile Ala Met Arg Ser Leu Thr Ala Ala Val Thr Ala Ala 225 230 235
240 Gly Gly Asn Ala Thr Tyr Val Leu Asp Gly Val Pro Arg Met Arg Glu
245 250 255 Arg Pro Ile Gly Asp Leu Val Val Gly Leu Lys Gln Leu Gly
Ala Asp 260 265 270 Val Asp Cys Phe Leu Gly Thr Asp Cys Pro Pro Val
Arg Val Asn Gly 275 280 285 Ile Gly Gly Leu Pro Gly Gly Lys Val Lys
Leu Ser Gly Ser Ile Ser 290 295 300 Ser Gln Tyr Leu Ser Ala Leu Leu
Met Ala Ala Pro Leu Ala Leu Gly 305 310 315 320 Asp Val Glu Ile Glu
Ile Ile Asp Lys Leu Ile Ser Ile Pro Tyr Val 325 330 335 Glu Met Thr
Leu Arg Leu Met Glu Arg Phe Gly Val Lys Ala Glu His 340 345 350 Ser
Asp Ser Trp Asp Arg Phe Tyr Ile Lys Gly Gly Gln Lys Tyr Lys 355 360
365 Ser Pro Lys Asn Ala Tyr Val Glu Gly Asp Ala Ser Ser Ala Ser Tyr
370 375 380 Phe Leu Ala Gly Ala Ala Ile Thr Gly Gly Thr Val Thr Val
Glu Gly 385 390 395 400 Cys Gly Thr Thr Ser Leu Gln Gly Asp Val Lys
Phe Ala Glu Val Leu 405 410 415 Glu Met Met Gly Ala Lys Val Thr Trp
Thr Glu Thr Ser Val Thr Val 420 425 430 Thr Gly Pro Pro Arg Glu Pro
Phe Gly Arg Lys His Leu Lys Ala Ile 435 440 445 Asp Val Asn Met Asn
Lys Met Pro Asp Val Ala Met Thr Leu Ala Val 450 455 460 Val Ala Leu
Phe Ala Asp Gly Pro Thr Ala Ile Arg Asp Val Ala Ser 465 470 475 480
Trp Arg Val Lys Glu Thr Glu Arg Met Val Ala Ile Arg Thr Glu Leu 485
490 495 Thr Lys Leu Gly Ala Ser Val Glu Glu Gly Pro Asp Tyr Cys Ile
Ile 500 505 510 Thr Pro Pro Glu Lys Leu Asn Val Thr Ala Ile Asp Thr
Tyr Asp Asp 515 520 525 His Arg Met Ala Met Ala Phe Ser Leu Ala Ala
Cys Ala Glu Val Pro 530 535 540 Val Thr Ile Arg Asp Pro Gly Cys Thr
Arg Lys Thr Phe Pro Asp Tyr 545 550 555 560 Phe Asp Val Leu Ser Thr
Phe Val Lys Asn 565 570
* * * * *