# Patent

## Title
Method for plant breeding

## 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.

## Background
This application is a continuation of application Ser. No. 10/869,324, filed Jun. 16, 2004, now abandoned; which is a continuation of application Ser. No. 09/698,789, filed Oct. 27, 2000, now issued as U.S. Pat. No. 6,762,344; which is a continuation of application Ser. No. 08/927,368 filed Sep. 11, 1997, now abandoned, which is a continuation-in-part of application Ser. No. 08/899,247, filed Jul. 23, 1997, now abandoned; which is a continuation-in-part of Application Ser. No. 08/832,078 filed Apr. 3, 1997, now issued as U.S. Pat. No. 6,040,497.


BACKGROUND OF THE INVENTION
1. Field of the Invention
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.
2. Description of the Related Art
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
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.
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.
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).
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.
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.
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).
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
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.
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 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.
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.
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.
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.
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 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.
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 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.
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.
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.
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.
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.
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.
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.
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.
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×, 2×, 3× or 4× (1× is equivalent to 16 ounces of RoundupTM 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.
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×, 2× or 4×.
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×, 2×, or 4×.
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×, 2× or 4×.
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.
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.
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.
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.
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.
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.
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.
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.
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
FIG. 1. Plasmid map of pDPG165. Restriction sites are shown and locations are indicated in base pairs.
FIG. 2. Plasmid map of pDPG427. Restriction sites used for Southern blot analyses are shown and locations are indicated in base pairs.
FIG. 3. Plasmid map of pDPG434. Restriction sites used for Southern blot analyses are shown and locations are indicated in base pairs.
FIG. 4. Plasmid map of pDPG434. Restriction sites used for Southern blot analyses are shown and locations are indicated in base pairs.
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 EcoRY. 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 LPSPS gene.
FIG. 6. Southern blot analysis to estimate the copy number and integrity of the mutant EPSPS Gene. Lane 1 contains GA21 DNA digested with EoRI/XbaI. Lane 2 contains nontransformed control DNA digested with EcoRI/XbaI. Lane 3 contains pDPG434 digested with LcoRI/XbaI. The blot was probed with the 324 bp LPSPS gene PCR fragment.
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.
FIGS. 8A and 8B. Effect of glyphosate application on the growth and fertility of DK580 and DK626 BC.sub.4 hybrids of GA21, FI117, GG25 and GJ11 transformation events. Treatments consisted of glyphosate applications at the 0×, 1× and 4× rates (1×=16 ounces of ROUNDUP ULTRATM/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.
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.
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′ polyadenylation 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.
FIGS. 11A, 11B, and 11C. Southern blot analysis to detect transgene insertions GA21, GG25 and GJ11 using various restriction enzymes. Genomic DNA of a nontransformed 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).
FIG. 12. 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 (1-4), hybrid (DK580 or DK626), transformation event (GA21, FI117, GG25, or GJ11), transformed or non-transformed status (N or T), glyphosate application level (0×, 1× or 4×), 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.
FIG. 13. Plasmid map of pDPG425. Major components and restriction sites are shown and locations are indicated in kilobase pairs.
FIG. 14. Plasmid map of pDPG405. Major components and restriction sites are shown and locations are indicated in base pairs.



DETAILED DESCRIPTION OF THE INVENTION
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:

    
    
        (a) plant seeds of the first (starting line) and second (donor plant line that comprises a mutant EPSPS gene) parent plants;
        (b) grow the seeds of the first and second parent plants into plants that bear flowers;
        (c) pollinate the female flower of the first parent plant with the pollen of the second parent plant; and
        (d) harvest seeds produced on the parent plant bearing the female flower.

Backcross conversion is herein defined as the process including the steps of:

        (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;
        (b) selecting one or more progeny plant containing the desired gene, DNA sequence or element;
        (c) crossing the progeny plant to a plant of the second genotype; and
        (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.
    
    


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.
It is contemplated that glyphosate resistant plants may be obtained by transfer of the DNA sequence comprising a mutant EPSPIS 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.
I. Herbicide Control of Weeds
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.
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:

    
    
        1. Competing with crop plants for the essentials of growth and development.
        2. Production of toxic or irritant chemicals that cause human or animal health problems.
        3. Production of immense quantities of seed or vegetative reproductive parts or both that contaminate agricultural products and perpetuate the species in agricultural lands.
        4. Production on agricultural and nonagricultural lands of vast amounts of vegetation that must be disposed of.
    
    


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.
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.
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 (Solnaceae) 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.
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.
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 m 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.
A diverse group of weed species necessitates a range of weed control methods in corn. Broad leaf weeds such as velvedeaf, 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.







TABLE 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.






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.
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).
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.
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.
II. DNA Delivery
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.
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.
(i) Electroporation
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.
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.
(ii) Microprojectile Bombardment
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.
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.
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 microprojectile 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.
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.
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.
III. Recipient Cells for Transformation
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.
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.
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.
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. Medistematic cells (i.e., 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.
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.
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.
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.
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).
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.
(i) Culturing Cells to be Recipients for Transformation
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.







TABLE 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



BASAL
SU-

COMPONENTS**


MEDIA 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






10−7M 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






10−7M ABA






200 mg myo-inositol






Bactoagar


177
MS
  3%
6.0
MS vitamins






.25 mg 2,4-D






10 mg BAP






10−7M 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 AgNO3






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 AgNO3






Gelgro


401
MS
  3%
6.0
3.73 mg Na2EDTA






.25 mg thiamine






1 mg 2,4-D






2 mg NAA






200 mg casein hydrolysate






500 mg K2SO4






400 mg KH2PO4






100 mg myo-inositol


402
MS
  3%
6.0
3.73 mg Na2EDTA






.25 mg thiamine






1 mg 2,4-D






200 mg casein hydrolysate






2.9 g L-proline






500 mg K2SO4






400 mg KH2PO4






100 mg myo-inositol


409
MS
  3%
6.0
3.73 mg Na2EDTA






.25 mg thiamine






9.9 mg dicamba






200 mg casein hydrolysate






2.9 g L-proline






500 mg K2SO4






400 mg KH2PO4






100 mg myo-inositol


501
Clark's
  2%
5.7



Medium***


607
1/2 × 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 × 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 AgNO3






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 MgCl2






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 AgNO3






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 NH4NO3 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.






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:

    
    
        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.
        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.
        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.
        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.
    
    


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.







TABLE 3







Initiated Maize Cultures











Genotype
Culture
Method
Transformable
Fertile Plants





A188 × B73
G(1 × 6)92
1
+
−



G(1 × 6)716
1, 2
+
+



G(1 × 6)82
1
+
+



G(1 × 6)98
1
−
NA



G(1 × 6)99
1
−
NA



D(1 × 6)122#3
2
−
NA



D(1 × 6)114
2
−
NA



D(1 × 6)17#33
2
−
NA



HB13-3
3
+
+



HA133-227
2
−
NA



G(6 × 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 × B84
G(1 × M)82
1
+
−


A188 × H99
HJ11-7
3
+
−


B73 × A188
G(6 × 1)12#7
2
−
NA



D(6 × 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






(ii) Media
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.
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.
(iii) Maintenance
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.
(iv) Cryopreservation Methods
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.
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° C.) concentrated cryoprotectant mixture stepwise over a period of one to two hours to pre-cooled (0° C.) cells. The mixture was maintained at 0° 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° 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° C./minute to −8° C. and held at this temperature for ice nucleation.
Once extracellular ice formation had been visually confirmed, the tubes were cooled at 0.5° C./minute from −8° C. to −35° 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° 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.
Thawing of the extracellular solution was accomplished by removing the cryo-tube from liquid nitrogen and swirling it in sterile 42° 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.
IV. DNA Segments Comprising Exogenous Genes
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.
The plants of the current invention have a mutant EPSPS gene which confers glyphosate resistance. The preferred EPSPS sequence, as shown in SEQ ID NO:5, 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.
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).
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.







TABLE 4







Vectors used in the transformation of maize glyphosate


resistant lines GA21, GG25, GJ11, and FI117













DELIBERATE


RECOMBINANT VECTOR
PARENT
INSERT
EXPRESSION


DESIGNATION & SOURCE
REPLICON
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









KEY: Insert DNA and Deliberate Expression Attempt

    1. The bar gene from Streptomyces hygroscopius 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.
    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.
    3. Promoter sequences from the Cauliflower Mosaic Virus genome. Odell, J. T., Nagy, F., and Chua, N. -H. 1985. Nature 313: 810-812.
    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.
    5. Enhancer sequences from the maize alcohol dehydrogenase gene. Callis, J., Fromm, M. E., Walbot, V., 1987. Genes Dev. 1: 1183-1200.
    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.
    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).
    8. Fused promoter sequences from Cauliflower Mosaic Virus genome and Arabidopsis thaliana H4 histone gene. Constructed by Rhone Poulenc Agrochimie.
    9. Actin-1 5′ region including promoter from Oryza sativa (McElroy et al. 1991).









TABLE 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 gene containing the promoter and


promoter and

first intron (McElroy et al., 1991)


intron


optimized
0.37
chloroplast transit peptide sequence constructed based on transit


transit peptide

peptide sequences from maize and sunflower ribulose-1,5-bis


(OTP)

phosphate carboxylase oxygenase (RuBisCo) genes (Lebrun et




al., 1996)


mutant maize
1.34
wild-type maize EPSPS gene (Lebrun et al., 1991) containing


EPSPS gene

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 β-galactosidase or lacZ protein (Yanisch-




Perron et al., 1985)


bla
0.86
The TEM type β-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)









V. Identification of Transformed Cells Using Selection

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.
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 LibertyTM 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.
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.
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.
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.
VI. Production and Characterization of Stable Transgenic Corn
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.
(i) Selection
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.
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 mM 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.
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.
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.
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.
(ii) Regeneration and Seed Production
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 conducive 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.
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 CO2, and 25-250 microeinsteins m−2 s−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° 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.
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 10−5M abscisic acid and then transferred to growth regulator-free medium for germination.
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 (RO) and their progeny (R1) 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.
(iii) Characterization
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.
1. DNA Integration, RNA Expression and Inheritance
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.
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.
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.
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.
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.
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.
2. Gene Expression
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.
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.
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 14C-acetyl CoA or for anthranilate synthase activity by following loss of fluorescence of anthranilate, to name two.
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.
VII. Purification of Proteins
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.).
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.
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.
VIII. Genetic Analysis of Glyphosate Resistant Transgenic Plants
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.
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.
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).
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.
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.
(i) Primers and Probes
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.
In preferred embodiments, the probes or primers are labeled with radioactive species (32P, 14C, 35S, 3H, or other label), with a fluorophore (rhodamine, fluorescein), an antigen (biotin, streptavidin, digoxigenin), or a chemillumiscent (luciferase).
(ii) Template Dependent Amplification Methods
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 PCRTM) 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.
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.
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.
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.
Qbeta Replicase, described in PCT Application No. PCT/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.
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).
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.
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.
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.
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 I), 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.
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, i.e., 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).
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.
(iii) Southern/Northern Blotting
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.
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.
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.
(iv) Separation Methods
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.
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).
(v) Detection Methods
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.
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.
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.
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.
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.
(vi) Design and Theoretical Considerations for Relative Quantitative RT-PCR
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.
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.
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.
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.
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.
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.
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.
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.
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.
(vii) Chip Technologies
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).
IX. Regeneration of Plants From Transformed Cells
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.
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.
X. Glyphosate Induced Male-Sterility in GJ11 and GG25
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 ULTRATM). 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.
(i) Utilization of Herbicide Inducible Male-sterility in Breeding Programs
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.
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.
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.
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.
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.
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.
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 F1 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.
In one embodiment, corn seed prepared by such a process is a first generation seed capable of being grown into an F1 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.
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 (F1) corn hybrid plant is produced. This F1 seed, the F1 hybrid corn plants grown therefrom, and seed of that F1 hybrid corn plant are contemplated as aspects of the present invention. The goal of a process of producing an F1 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 F1 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:

    
    
        (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;
        (b) cultivating or growing the seeds of the first and second parent corn plants;
        (c) applying 8 to 96 ounces per acre of glyphosate (ROUNDUP ULTRATM) to the parent corn plants between the V8 and VT stages of development;
        (d) allowing cross-pollination to occur between the first and second parent corn plant;
        (e) harvesting seeds produced on the first plant; and, where desired,
        (f) growing the harvested seed into a corn plant.
    
    


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.
(ii) Use of Herbicide Applications for Seed Purity
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 gray 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.
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.
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.
(iii) Applicability of Herbicide Induced Male-sterility
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 GG25 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.
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.
XL. Definitions
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.
Inviable Pollen: pollen which is not capable of fertilizing a plant to produce seed.
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.
Male-Sterile: a male-sterile plant is one which is not capable of self fertilization or fertilization of other plants to produce seeds.
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.
XII. Deposit Information
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. §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
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 
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 μM 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.
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.
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
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 Ser. 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.
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).
DNA was precipitated onto gold particles as follows. A stock solution of gold particles was prepared by adding 60 mg of 0.7 μm or 1 μm gold particles to 1000 μl absolute ethanol and incubating for at least 3 hours at room temperature followed by storage at −20° C. Twenty to thirty five μ1 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 μl of DNA solution containing about 10-20 μg of the NotI restricted pDPG434 mutant EPSPS expression cassette. Two hundred twenty microliters sterile water, 250 μl 2.5 M CaCl2 and 50 μl spermidine were added. The mixture was thoroughly mixed and placed on ice, followed by vortexing at 4C for 10 minutes and centrifugation at 500 rpm for 5 minutes. The supernatant was removed and the pellet resuspended in 600 μl absolute ethanol. Following centrifugation at 500 rpm for 5 minutes the pellet was resuspended in 36 μl of absolute ethanol and was allowed to settle for 4 minutes. Ten μl 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.
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 100×20 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
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 μm 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° 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.
Linearized plasmid DNA, about 60 μg of NotI excised EPSPS expression cassette from pDPG427 (GG25) or pDPG443 (GJ11); or 100 μg of whole pDPG165 and pDPG434 (FI117) plasmid DNA (50 μg 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 60×15 mm petri dish. Cells were electroporated with a 70, 100, or 140 volt (V) pulse discharged from a 140 microfarad (μf) capacitor.
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
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° C. Transformed plants that developed shoots roots were transferred to soil.
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
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 100×20 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
Plants regenerated from callus lines GA21, GG25, GJ11, and FI117 (R0 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 R0 crosses (R1 generation) in a greenhouse by application of RoundupTM 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
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′ (SEQ ID NO:1). The sequence of the lower primer located in nos is 5′-GCAAGACCGGCAACAGGATTC-3′ (SEQ ID NO:2). 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
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 ′-TTTGGCTCTTGGGGATGTG-3′ (upper) (SEQ ID NO:3) and 5′-TTACGCTAGTCTCGGTCCAT-3′ (lower) (SEQ ID NO:4). Probes were labeled with 32P using the random priming method (Boebringer Mannheim) and purified using Quik-Sep® spin columns (Isolab Inc., Akron, Ohio). Blots were prehybridized at 65° C. for 1-2 hours and hybridized with denatured probe for approximately 18 hours at 65° C. Prehybridization and hybridization solution consisted of 5×SCP, 2× Denhardt's Solution, 0.05 M Tris, pH 8.0, 0.2 % SDS, 10 mM EDTA, 100 mg/l dextran sulfate, and 125 μg/ml denatured salmon sperm DNA. Following hybridization, blots were washed 4 times for 10 mm. in 0.25×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 mm. followed by neutralization in 0.2 M Tris, pH 7.5/0.2% SDS/0.1 ×SCP for 20 minutes at approximately 25° C.
Approximately 10 μg 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° C.
Example 9 
Copy Number and Integrity of the Mutant EPSPS Transgene in GA21 
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.
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.
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 
To confirm the lack of plasmid backbone sequences containing the ColE1 origin of replication and the bla gene encoding β-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 pDPG165, pDPG434 and pDPG443 
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 pIJ4101. 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.).
The plasmids pDPG434 (FIG. 3) and pDPG443 (FIG. 4) were constructed by cloning the respective promoters into SmaI-linearized pDPG425 (FIG. 13). 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 2×35S/Arabidopsis histone promoter was isolated as a 1.8 kb EcoRI/HindIII fragment from pDPG405 (FIG. 14). The above mentioned promoter fragments were T4 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 
BC4 hybrids of DK580 and DK626 were produced containing one of the FI117, 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× glyphosate (i.e. water only), 1× glyphosate, or 4× glyphosate (the 1× level corresponds to 16 ounces/acre of ROUNDUP ULTRATM).
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 (SEQ ID NO:5). 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, per cent grain moisture at harvest, and test weight.
The results show that all 4 transformation events gave significant resistance to glyphosate at both the 1× and 4× application levels (FIGS. 7A, 7B). Overall, the GA21 transformation event yielded the most efficacious resistance, in that at the 4× 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
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. 13. 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.
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.
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× 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
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, G625, GJ11, and/or FI117 transformation event.
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.
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× 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
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, FI117 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.
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.
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. Freeze-dried 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 NaCI, 0.05 M Tris-HCI ph 8.0, 0.01 M EDTA, 1% sircosine). Tissue/buffer homogenate is extracted with 3 ml phenol/chloroform. The aqueous phase is separated by centrifugation, and precipitated twice using 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 μl TE (0.01 M Tris-HCI, 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×SCP (20 SCP: 2M NaCl, 0.6 M disodium phosphate, 0.02 M disodium EDTA).
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.
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×SCP, 10% dextran sulfate, 2% sarcosine, and 500 μg/ml denatured salmon sperm DNA and 32P-labeled probe generated by random priming (Feinberg & Vogelstein, 1983). Hybridized filters are washed in 2×SCP, 1% SDS at 65° 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.
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
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 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.
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 MgCl2, 50 mM KCl, 0.1 mg/ml gelatin, 200 μM each dATP, dCTP, dGTP, dTTP, 0.5 μM each forward and reverse DNA primers, 20% glycerol, and 2.5 units Taq DNA polymerase. The primer sequences are (upper) 5′-TTTGGCTCTTGGGGATGTG-3′ (SEQ ID NO:3) and (lower)5′-TTACGCTAGTCTCGGTCCAT-3′ (SEQ ID NO:4). 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 μl 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.
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×SCP (20×SCP: 2 M NaCl, 0.6 M disodium phosphate, 0.02 M disodium EDTA). Filters were prehybridized at 65° C. in 6×SCP, 10% dextran sulfate, 2% sarcosine, and 500 μg/ml heparin (Chomet et al., 1987) for 15 min. Filters were hybridized overnight at 65 C in 6×SCP containing 100 μg/ml denatured salmon sperm DNA and 32P-labeled probe. Filters were washed in 2×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 H2O to remove the first probe and then prehybridized as described above.
Example 17 
Weed Control in Agricultural Fields of Glyphosate Resistant Maize Plants
RoundupTM is a commercial formulation of glyphosate manufactured and sold by the Monsanto Company. The amount of RoundupTM (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 RoundupTM to 256 ounces RoundupTM per acre (the 1× rate is equivalent to 16 ounces per acre of RoundupTM, i.e., 64 ounces/acre is 4×). Preferably, from 8 ounces to 128 ounces per acre of RoundupTM 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 RoundupTM may be applied to the field. An application of RoundupTM in excess of the 1× rate, including 1×, 2×, 3×, 4× 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.
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× 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× 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).
A farmer may also apply a combination of herbicides including RoundupTM, 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 RoundupTM in order to complement the activity of RoundupTM and thereby increase the efficiency of weed control. For example, RoundupTM 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 RoundupTM 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 RoundupTM.
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. RoundupTM 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, RoundupTM 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 RoundupTM may be desired in order to control weed growth. For example, RoundupTM 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, RoundupTM may be applied on an as needed basis in order to control growth of particular weeds when required.
Example 18 
Utilization of Transgenic Crops
The ultimate goal in plant transformation is to produce plants which are useful to man. In this resect, 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.
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 Ramstad (1987).
References
The references listed below are incorporated herein by reference to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and/or compositions employed herein.

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## Claims

1. A method of plant breeding comprising the steps of:
(a) planting a population of plants comprising at least a first or second transgene that confers vegetative herbicide tolerance and female reproductive herbicide tolerance to a preselected glyphosate treatment, wherein the population comprises the first and second transgenes, and wherein the first transgene but not the second transgene confers male reproductive herbicide tolerance to the preselected glyphosate treatment;
(b) treating the population of plants with the preselected glyphosate treatment to render pollen that lacks the first transgene inviable; and
(c) allowing pollination to occur to produce at least a first progeny comprising the first transgene.

2. The method of claim 1, wherein the preselected glyphosate treatment comprises an application of from 8 ounces per acre to 96 ounces per acre of glyphosate.

3. The method of claim 1, wherein treating the population of plants with the preselected glyphosate treatment comprises an over-the-top application of glyphosate.

4. The method of claim 1 wherein the plants are inbred.

5. The method of claim 1 wherein the plants are hybrid.

6. The method of claim 1, wherein said first or second transgene comprises a mutant EPSPS gene operably linked to a promoter functional in said plants.