Methods for weed control using a transgenic seed comprising a nucleic acid encoding a dicamba-degrading enzymatic activity

CA 02653739 2013-05-10

METHODS FOR WEED CONTROL USING A TRANSGENIC SEED COMPRISING A NUCLEIC ACID ENCODING A DICAMBA-DEGRADING ENZYMATIC ACTIVITY BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to the field of weed management. More specifically, the invention relates to methods for using auxin-like herbicides such as dicamba for controlling weeds.

2. Description of the Related Art

Weeds cost farmers billions of dollars annually in crop losses and the expense of efforts to keep weeds under control. Weeds also serve as hosts for crop diseases and insect pests. The losses caused by weeds in agricultural production environments include decreases in crop yield, reduced crop quality, increased irrigation costs, increased harvesting costs, decreased land value, injury to livestock, and crop damage from insects and diseases harbored by the weeds. 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 problem, 3) production of immense quantities of seed or vegetative reproductive parts or both that contaminate agricultural products and perpetuate the species in agricultural lands, and 4) production on agricultural and nonagricultural lands of vast amounts of vegetation that must be disposed of. The damage caused can be significant. For example, it is estimated that between 1972 and 1976 com yields were reduced by about 10% due to weeds (Chandler, 1981).

Among weeds that serve as hosts for crop pests, for example, pepperweed and tansymustard (Descurainia sp.) maintain large populations of diamondback moths during the late fall, wmter, and spring. They are also hosts to the turnip aphid and

CA 02653739 2008-11-28

WO 2007/143690

PCT/US2007/070517 green peach aphid. Several weed species of the nightshade family (Solanaceae) are hosts to insects that commonly attack eggplant, pepper, potato, and tomato. For example, horsenettle (Solarium 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.

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.

Chemical herbicides have provided an effective method of weed control over the years. Herbicides can generally be applied pre-emergence and/or post-emergence. Pre-emergence herbicides are applied in a field before a crop emerges from the soil. Such applications are typically applied to the soil before, at the same time, or soon after planting the crop. Such applications may kill weeds that are growing in the field prior to the emergence of the crop, and may also prevent or reduce germination of weeds that are present in the soil. Post-emergence herbicides are typically used to kill weeds after a crop has emerged in the field. Such applications may kill weeds in the field and prevent or reduce future weed germination. In either case, the herbicides may be applied to the surface of the soil, mixed with the soil, over the top of the plant, or applied by any other method known to those of skill in the art.

One weed control strategy is to apply an herbicide such as dicamba to a field before sowing seeds. However, after applying the herbicide to a field, a farmer has to wait at least several weeks before sowing the field with crop seeds such that the herbicide has killed most of the weeds and has degraded so as not injure the sown crop. For example, plants are especially sensitive to dicamba and it has been

CA 02653739 2013-12-18 recommended that dicamba formulations such as Banvel™ or Sterling™ be applied 30 days prior to planting for controlling weeds. A comprehensive list of weeds that are controlled by dicamba is available (Anonymous, 2007). The herbicide is particularly useful for control of taller weeds and more difficult to control weeds such as purslane, sicklepod, mominglory and wild buckwheat. Dicamba can be used to control weeds not susceptible to other herbicides. Following the application of Clarity™, another formulation of dicamba, a minimum accumulation of one inch of rainfall or overhead irrigation followed by a 14 day waiting period for the 4 to 8 ounce/acre rates or a 28 day waiting period for the 16 ounce/acre rates has been recommend for controlling weeds in a soybean field (see Table 22 in VanGessel and Majek, 2005). Also, the Clarity® label recommends that it be applied at least 15 days prior to sorghum planting. Similarly, for cotton, a waiting period of 21 days is recommended after applying Clarity® or Banvel® to the field, before planting the cotton seeds (Craig et,al., 2005, Crop Profile for Cotton (Gossypium hirsutum) in Tennessee IPM Center) and no preemergence and post-emergence application are recommended. The waiting period is also dependent on the crop growing environment at any give time, such as the type of soil (soil having organic activity will degrade dicamba faster), moisture content, rainfall, temperature, as well as type of formulation and rate of application.

The herbicide 2,4-D has been recommended for controlling certain weeds in a soybean field such as mustard spp., plantains, marestail, and 2,4-D susceptible annual broadleaf weeds by applying it 7 to 30 days prior to planting, depending on rate and formulation (ester or amine) (see Table 22 in VanGessel and Majek, 2005).

One method that has been successfully used to manage weeds combines herbicide treatments with crops that are tolerant to the herbicides. In this manner, herbicides that would normally injure a crop can be applied before and during growth of the crop without causing damage. Thus, weeds may be effectively controlled and new weed control options are made available to the grower. In recent years, crops tolerant to several herbicides have been developed. For example, crops tolerant to 2,4-dichlorophenoxyacetic acid (Streber and Willmitzer, 1989), bromoxynil (Stalker et al., 1988), glyphosate (Cornai et al., 1985) and phosphinothricin (De Block et al., 1987) have been developed.

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517

Recently, a gene for dicamba monooxygenase (DMO) was isolated from Pseudomonas maltophilia (US Patent Application No: 20030135879) which is involved in the conversion of a herbicidal form of the herbicide dicamba (3,6dichloro-o-anisic acid) to a non-toxic 3,6-dichlorosalicylic acid. The inventors 5 reported the transformation of the DMO gene into tobacco and Arabidopsis. The transformed plant tissue was selected on kanamycin and regenerated into a plant. However, herbicide tolerance was not demonstrated or suggested in immature tissues or seedlings or in other plants. Pre-emergence herbicide tolerance to dicamba was not described. Transgenic soybean plants and other plants tolerant to application of 10 dicamba are described in Behrens et al. (2007).

Dicamba is one member of a class of herbicides commonly referred to as “auxin-like” herbicides or “synthetic auxins.” These herbicides mimic or act like the natural plant growth regulators called auxins. Auxin-like herbicides appear to affect cell wall plasticity and nucleic acid metabolism, which can lead to uncontrolled cell 15 division and growth. The injury symptoms caused by auxin-like herbicides include epinastic bending and twisting of stems and petioles, leaf cupping and curling, and abnormal leaf shape and venation.

Dicamba is one of the many auxin-like herbicides that is a low-cost, environmentally-friendly herbicide that has been used as a pre-emergence herbicide 20 (i.e., 30 days prior to planting) in dicots and as a pre- and/or post-emergence herbicide in com, sorghum, small grains, pasture, hay, rangeland, sugarcane, asparagus, turf, and grass seed crops to effectively control annual and perennial broadleaf weeds and several grassy weeds (Crop Protection Chemicals Reference, 1995). Unfortunately, dicamba can injure many commercial crops including beans, soybeans, cotton, peas, 25 potatoes, sunflowers, tomatoes, tobacco, and fruit trees, ornamental plants and trees, and other broadleaf plants when it comes into contact with them. Soybean and cotton are particularly sensitive to dicamba. Thus, applications of dicamba must generally occur several weeks before planting of sensitive crops to ensure that residual dicamba is sufficiently cleared from the crop environment before crops emerge. For post30 emergent weed control in com, dicamba is the 5th most widely used herbicide for broad leaf weeds. However, although the optimal rate for broad leaf weed control is between 280 to 560 g/h (grams/hectare), the average use rate in com is 168 g/h as at higher use rates and under certain environmental conditions, dicamba can injure com.

CA 02653739 2008-11-28

WO 2007/143690

PCT/US2007/070517

As noted above, current manufacturer’s guidelines typically require at least a 30 day delay between the application of dicamba and the planting of sensitive crops. This inability to apply dicamba close to the time that crops are planted delays sowing time and shortens the growing season, thereby increasing the risk of exposing crops to frost in the fall. The delay also means that the farmers have to go through the field twice; once for planting and once for spraying, thereby increasing fuel and wear-tear costs to the farmers. Improvements over the state of the art that would eliminate the delay would positively impact the quality and quantity of the crop which could result and reduce economic losses to farmers. More effective weed control would also reduce the risk of weeds developing resistance to existing herbicides.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for controlling weed growth in a field comprising: a) applying a herbicidally effective amount of an auxin-like herbicide to a crop-growing environment; and planting a transgenic seed of a dicotyledonous plant expressing a nucleic acid encoding dicamba monooxygenase in soil of the crop-growing environment, wherein the seed germinates within 30 days or less of applying the herbicide and wherein the dicamba monooxygenase comprises at least 70% sequence identity to the polypeptide sequence of SEQ ID NO:2; and c) allowing the seed to germinate into a plant. In certain embodiments, the seed germinates within four weeks, three weeks, two weeks, or less than one week after treating the growing environment with the auxin-like herbicide. The treated growing environment may be, for example, a field in which a crop is planted. A population of seeds of a plant tolerant to the auxin-like herbicide may be planted in the field. Treating the environment can be carried out according to known techniques in the art using, for example, commercially available formulations of auxin-like herbicides such as dicamba. The environment includes an area for which control of weeds is desired and in which the seed of a plant tolerant to the auxin-like herbicide can be planted. A weed can be directly contacted with herbicide in the environment and soil in the environment can be contacted with the herbicide, preventing or reducing weed growth in the soil. The step of treating the environment with a herbicide may be carried out before, after, or concurrently with the step of planting the soil with the transgenic seed. The transgenic seed may be planted into soil in the environment, for example,

<img file="CA2653739C_D0001.tif" />

CA 02653739 2013-05-10 within three weeks before or after treatment, including from between about two weeks, one week and less than 1 week before or after treatment, further including from between about 1, 2, 3, 4, 5, or 6 days before or after treatment, including concurrently with treatment. In the method, the seed may germinate, for example, from between about 30 days and 0 days after treating the environment, including between about 21, 18, 16, 14, 12, 10, 8, 6, 5, 4, 3, 2, 1 and about 0 days after treating the environment. The method may further comprise applying one or more additional treatments of an auxin-like herbicide after the seed germinates and/or the plant is growing. In certain embodiments, a second treatment is carried out at a time selected from the group consisting of between about the 1 to 2 leaf and 3 to 4 leaf stages, before flowering, at flowering, after flowering, and at seed formation. In one embodiment, the second treatment comprises applying dicamba and/or a 2,4-dichlorophenoxyacetic compound (2,4-D).

In a method of the invention, the auxin-like herbicide may be selected from the group consisting of a phenoxy carboxylic acid compound, benzoic acid compound, pyridine carboxylic acid compound, quinoline carboxylic acid compound, and benazolinethyl compound. Examples of a phenoxy carboxylic acid compound include 2,4-dichlorophenoxyacetic acid and (4-chloro-2-methylphenoxy) acetic acid. In certain embodiments, a herbicidally effective amount of 2,4-D and/or (4-chloro-2methylphenoxy) acetic acid used is between about 2 g/ha (grams/hectare) to about 5000 g/ha, including about 50 g/ha to about 2500 g/ha, about 60 g/ha to about 2000 g/ha, about 100 g/ha to about 2000 g/ha, about 75 g/ha to about 1000 g/ha, about 100 g/ha to about 500 g/ha, and from about 100 g/ha to about 280 g/ha. In one embodiment found to function particularly well with the invention, dicamba is used as the herbicide. In certain embodiments, a herbicidally effective amount of dicamba used may be from about 2.5 g/ha to about 10,080 g/ha, including about 2.5 g/ha to about 5,040 g/ha, about 5 g/ha to about 2,020 g/ha, about 10 g/a to about 820 g/h and about 50 g/ha to about 1,000 g/ha, about 100 g/ha to about 800 g/ha and about 250 g/ha to about 800 g/ha.

In a method of the invention a plant may be used exhibiting tolerance to auxinlike herbicides including dicamba. Such a plant may comprise a nucleic acid encoding a dicamba monooxygenase. In one embodiment, the plant is defined as comprising a nucleic acid encoding a dicamba monooxygenase that has at least 70%

CA 02653739 2008-11-28

WO 2007/143690

PCT/US2007/070517 identity to a polypeptide sequence of any one or more of SEQ ID NOs:2, 4, 6, 8, 10 or 12, including at least about 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% and greater sequence identity to these sequences. Polypeptide or polynucleotide comparisons may be carried out and identity determined as is known in the art, for example, using MEGAlign (DNAStar, Inc., 1228 S. Park St., Madison, Wis. 53715) with default parameters. Such software matches similar sequences by assigning degrees of similarity or identity.

The methods of the invention may be used in connection with plants that exhibit susceptibility to auxin-like herbicides such as dicotyledonous (dicot) plants. In certain embodiments, a dicotyledonous plant is used selected from the group consisting of alfalfa, beans, broccoli, cabbage, carrot, cauliflower, celery, cotton, cucumber, eggplant, lettuce, melon, pea, pepper, pumpkin, radish, rapeseed, spinach, soybean, squash, tomato, and watermelon. In some embodiments, the dicot is soybean, cotton, or canola.

In another aspect, the invention provides a method for controlling a weed in a field comprising: a) planting a transgenic seed in a field, wherein the seed comprises transgenes conferring tolerance to an auxin-like herbicide and a second herbicide; b) growing the seed into a plant; and c) treating the field with an amount of the auxinlike herbicide and the second herbicide in amounts effective to control weed growth. In some embodiments, the second herbicide may be glufosinate (De Block et al., 1987) , a sulfonylurea (Sathasiivan et al., 1990), an imidazolinone (U.S. 5,633,437; U.S. 6,613,963), bromoxynil (Stalker et al., 1988), dalapon or 2,2-Dichloropropionic acid (Buchanan-Wollaston et al., 1989), cyclohexanedione (U.S. 6,414,222) , a protoporphyrinogen oxidase inhibitor (U.S. 5,939,602), norflurazon (Misawa et al., 1993 and Misawa et al., 1994), or isoxaflutole (WO 96/38567) herbicide, among others. The auxin-like herbicide and the second herbicide may be applied simultaneously or separately. In a particular embodiment, the second herbicide is glyphosate and the auxin-like herbicide is dicamba. In one embodiment, the plant comprises a nucleic acid that has at least 70% sequence identity to a nucleic acid sequence of any one or more of SEQ ID NOs: 1, 3, 5, 7, 9, or 11, including at least about 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% and greater sequence identity to these sequence.

<img file="CA2653739C_D0002.tif" />

CA 02653739 2013-05-10

In further embodiments, a plant such as the foregoing is defined as comprising a transgene conferring glyphosate tolerance. Glyphosate resistant 5enolpyruvylshikimate-3-phosphate synthases (EPSPS) are well known in the art and disclosed, for example, in U.S. Patent 5,627,061, U.S. Patent 5,633,435, U.S. Patent 6,040,497, U.S. Patent 5,094,945, WO04074443, and W004009761. Nucleic acids encoding glyphosate degrading enzymes, for example, glyphosate oxidoreductase (GOX, U.S. Patent 5,463,175), and nucleic acids encoding glyphosate inactivating enzymes, such as glyphosate-N-acetyl transferase (GAT, U.S. Patent publication 20030083480; U.S. Patent Publication 20070079393) and glyphosate decarboxylase (W005003362 and US Patent Application 20040177399) are also known. In certain embodiments, the GAT enzyme comprises the sequence of GAT4601 (SEQ ID NO: 19), or is encoded by a transgene comprising the nucleic acid sequence of SEQ ID NO:18. In a particular embodiment, the GAT polypeptide is expressed using the SCP1 promoter.

In the method, treating the field may be carried out at a time selected from the group consisting of between about the 1 to 2 leaf and 3 to 4 leaf stages, before flowering, at flowering, after flowering, and at seed formation. Treating the field may further be defined as carried out at a time proximate to step a) such that the seed germinates while the auxin-like herbicide remains in the soil in an amount effective to control growth of the weed. In the method, treating the field may be carried out about three weeks, two weeks, 1 week or less than 1 week before step a). The auxin-like herbicide may be selected from the group consisting of a phenoxy carboxylic acid compound, benzoic acid compound, pyridine carboxylic acid compound, quinoline carboxylic acid compound, and benazolinethyl compound.

The phenoxy carboxylic acid compound may be selected from the group consisting of 2,4-dichlorophenoxyacetic acid, (4-chloro-2-methylphenoxy) acetic acid, and 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB). The amount of 2,4dichlorophenoxyacetic compound used may be lower than about 280 g/ha. The amount of 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB) used may be lower than about 1120 g/ha. The amount of (4-chloro-2-methylphenoxy) acetic acid compound used may be lower than about 280 g/ha. In one embodiment, the auxin like herbicide is dicamba. The amount of dicamba used may be, for example, from about 2.5 g/ha to about 10,080 g/ha, including about 2.5 g/ha to about 1040 g/ha, about 5 g/ha to about

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517

2040 g/ha, about 10 g/a to about 820 g/h, and about 50 g/ha to about 1000 g/ha. The amount of glyphosate may be from about 200 g/ha to about 1,600 g/h, including from about 200 g/ha to about 1,000 g/h, from about 200 g/ha to about 800 g/h, from about 200 g/ha to about 400 g/h, and from about 400 g/ha to about 800 g/h.

In yet another aspect, the invention provides a method for controlling weed growth in a crop-growing environment comprising: a) applying a herbicidally effective amount of an auxin-like herbicide to a crop-growing environment; b) planting a transgenic seed of a monocotyledonous plant comprising a nucleic acid encoding a dicamba degrading enzymatic activity, such as dicamba monooxygenase, 10 in soil of the crop-growing environment within 21 days of applying the auxin-like herbicide, wherein the herbicidally effective amount is an amount that does not damage the transgenic seed or a plant that germinates therefrom but will damage a seed or a plant that germinates therefrom of the same genotype that lacks the nucleic acid and is planted under the same conditions as the transgenic seed, wherein the 15 nucleic acid is selected from the group consisting of (1) a nucleic acid sequence encoding the polypeptide of SEQ ID NO:8, (2) a nucleic acid sequence comprising the sequence of SEQ ID NO:7, (3) a nucleic acid sequence that hybridizes to a complement of the nucleic acid sequence of SEQ ID NO:7 under conditions of 5X SSC, 50% formamide and 42°C, (4) a nucleic acid sequence having at least 70% 20 sequence identity to the nucleic acid sequence of SEQ ID NO:7, and (5) a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to the polypeptide sequence of SEQ ID NO:8; and c) allowing the seed to germinate into a plant. The nucleic acid sequence having at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:7 may encode a polypeptide comprising a cysteine 25 residue at position 112. This embodiment may combined with any of the methods and compositions provided above.

In particular embodiments of the invention, herbicide treatments to monocot plants may be made at higher rates and/or in closer proximity to emergence of crops than previously could be made without damaging crops. In specific embodiments, a 30 herbicidally effective amount of 2,4-D and/or MCPA, such as, for example, at least about 200, 300, 300, 500, 590, 650, 700, 800 or more g/ha of either or both herbicides, including from about 300 to about f200 g/ha, from about 500 to about f200 g/ha, from about 600 to about f200 g/ha, from about 590 to about f400 g/ha,

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517 and from about 700 to about 1100 g/ha of either or both herbicides. The herbicide may also be dicamba and the herbicidally effective amount may be, for example, at least about 168, 175, 190, 200, 225, 250, 280, 300, 400, 500, 560 or more g/ha of dicamba, including from about 200 g/ha to about 600 g/ha, from about 250 g/ha to 5 about 600 g/ha, from about 250 g/ha to about 800 g/ha, from about 225 g/ha to about

1120 g/ha, and from about 250 g/ha to about 1200 g/ha, from about 280 g/ha to about 1120 g/ha and from about 560 g/ha to about 1120 g/ha. In particular embodiment, the monocotyledonous plant is selected from the group consisting of com, rice, sorghum, wheat, rye, millet, sugarcane, oat, triticale, switchgrass, and turfgrass. Expressing the 10 transgenic dicamba-degrading enzymatic activity such as a monooxygenase, in a monocotyledonous crop plant, such as com, allows application of a higher level of dicamba to the crop for the purpose of weed control at any stage of plant growth, as compared to the level of dicamba that may be applied to a monocotyledonous crop plant that does not comprise a transgene that encodes such a dicamba-degrading 15 enzymatic activity.

In yet another aspect, the invention provides a method for controlling weed growth in a field comprising: a) applying a herbicidally effective amount of an auxinlike herbicide other than dicamba to a field, wherein the field comprises a transgenic dicotyledonous plant comprising a nucleic acid encoding dicamba monooxygenase or 20 is planted with a seed that germinates into said transgenic dicotyledonous plant within days of applying the herbicide, wherein the herbicidally effective amount is an amount that does not damage the transgenic dicotyledonous plant but will damage a plant of the same genotype that lacks the nucleic acid encoding dicamba monooxygenase, wherein the nucleic acid is selected from the group consisting of (1) 25 a nucleic acid sequence encoding the polypeptide of SEQ ID NO:8, (2) a nucleic acid sequence comprising the sequence of SEQ ID NO:7, (3) a nucleic acid sequence that hybridizes to a complement of the nucleic acid sequence of SEQ ID NO:7 under conditions of 5X SSC, 50% formamide and 42°C, (4) a nucleic acid sequence having at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:7, and (5) 30 a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to the polypeptide sequence of SEQ ID NO:8; and b) allowing the transgenic dicotyledonous plant to grow. In the method, step a) may comprise applying the herbicidally effective amount of an auxin-like herbicide to a growing environment

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517 adjacent to a growing environment comprising the transgenic dicotyledonous plant and allowing the herbicide to drift onto the plant or soil in which the plant grows. The auxin-like herbicide may be any herbicide as described herein. In the method, step b) may comprise allowing the transgenic dicotyledonous plant to grow to maturity. In 5 specific embodiments, the herbicidally effective amount may be defined as an amount that does not damage the transgenic plant.

In yet another aspect, the invention provides a method for increasing the efficiency of use of a herbicide delivery device comprising: a) obtaining a device that has been used to deliver a first composition comprising an auxin-like herbicide; and 10 b) delivering a second composition to the field using the device without first completely washing the device so that a herbicide residue comprising the auxin-like herbicide remains in the device and is delivered with the second composition to the field, wherein the field comprises a transgenic dicotyledonous plant expressing a nucleic acid encoding dicamba monooxygenase or is planted with a seed that 15 germinates into said transgenic dicotyledonous plant within 21 days of delivering the second composition, wherein the herbicide residue is present in an amount that does not damage the transgenic dicotyledonous plant but will damage a plant of the same genotype that lacks the nucleic acid encoding dicamba monooxygenase, wherein the nucleic acid is selected from the group consisting of (1) a nucleic acid sequence 20 encoding the polypeptide of SEQ ID NO:8, (2) a nucleic acid sequence comprising the sequence of SEQ ID NO:7, (3) a nucleic acid sequence that hybridizes to a complement of the nucleic acid sequence of SEQ ID NO:7 under conditions of 5X SSC, 50% formamide and 42°C, (4) a nucleic acid sequence having at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:7, and (5) a nucleic acid 25 sequence encoding a polypeptide having at least 70% sequence identity to the polypeptide sequence of SEQ ID NO:8.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates, in one aspect, to the unexpected discovery that preemergent applications of auxin-like herbicides such as dicamba may be made close to, 30 or even concurrently with the planting of crops. The invention provides superior weed control options, including reduction and/or prevention of herbicide tolerance in weeds. Pre-emergent applications of auxin-like herbicides such as dicamba have

CA 02653739 2013-05-10 previously required herbicide applications well in advance of planting and germination of plants susceptible to auxin-like herbicides to allow breakdown of the herbicide in the environment and avoid significant crop damage or death. Most crop plants, and particularly dicotyledonous plants such as soybeans and cotton are extremely sensitive to dicamba. Thus, the recommended post-application delays in planting by manufacturers must be closely followed.

Young plantlets and seeds are particularly sensitive to herbicides. Even in transgenic seeds and plants, immature tissues can insufficiently express the gene needed to render them tolerant to the herbicide, or may not have accumulated sufficient levels of the protein to confer tolerance. For example, mature plants have been found exhibiting high levels of tolerance to the herbicides Hamess™ (acetochlor), Lasso™ (alachlor), Trefian™ (Trifluralin), Eptam™ (EPTC), and/or Far-Go™ (triallate) but susceptibility to the herbicides at germination. As a result of this variability in young tissues, crop response to postemergence applications (e.g., in more mature vegetative tissues) of dicamba herbicides can significantly differ from the crop response to pre-emergent applications of herbicides in which younger more sensitive tissues are exposed. The former does not necessarily predict the latter. This is underscored in the case of plants highly sensitive to a given herbicide, such as dicots and the herbicide dicamba. Thus, the present invention unexpectedly shows that higher than predicted levels of crop safety can be achieved from pre-emergence applications of dicamba.

The present invention employs auxin-like herbicides, which are also called auxinic or growth regulator herbicides, or Group 4 herbicides (based on their mode of action). These types of herbicides mimic or act like the natural plant growth regulators called auxins. The action of auxinic herbicides appears to affect cell wall plasticity and nucleic acid metabolism, which can lead to uncontrolled cell division and growth.

Auxin-like herbicides include four chemical families: phenoxy, carboxylic acid (or pyridine), benzoic acid, and quinaline carboxylic acid. Phenoxv herbicides are most common and have been used as herbicides since the 1940s when (2,4dichlorophenoxy) acetic acid (2,4-0) was discovered. Other examples include 4-(2,4dichlorophenoxy) butyric acid (2,4-DB), 2-(2,4-dichlorophenoxy) propanoic acid (2,

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517

4-DP), (2,4,5-trichlorophenoxy)acetic acid (2,4,5-T), 2-(2,4,5-Trichlorophenoxy) Propionic Acid (2,4,5-TP), 2-(2,4-dichloro-3-methylphenoxy)-N-phenylpropanamide (clomeprop), (4-chloro-2-methylphenoxy) acetic acid (MCPA), 4-(4-chloro-otolyloxy) butyric acid (MCPB), and 2-(4-chloro-2-methylphenoxy) propanoic acid (MCPP).

The next largest chemical family is the carboxylic acid herbicides, also called pyridine herbicides. Examples include 3,6-dichloro-2-pyridinecarboxylic acid (Clopyralid), 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (picloram), (2,4,5trichlorophenoxy) acetic acid (triclopyr), and 4-amino-3,5-dichloro-6-fluoro-210 pyridyloxyacetic acid (fluroxypyr). Examples of benzoic acids include 3,6-dichloroo-anisic acid (dicamba) and 3-amino-2,5-dichlorobenzoic acid (choramben). Dicamba is a particularly useful herbicide for use in the present invention. A fourth chemical family of auxinic herbicides is the quinaline carboxylic acid family. Example includes 3,7-dichloro-8-quinolinecarboxylic acid (quinclorac). This 15 herbicide is unique in that it also will control some grass weeds, unlike the other auxin-like herbicides which essentially control only broadleaf or dicotyledonous plants. The other herbicide in this category is 7-chloro-3-methyl-8quinolinecarboxylic acid (quinmerac).

It was found, for example, that soybean plants transformed with dicamba 20 monooxygenase (DMO)-encoding polynucleotide constructs were tolerant to even early pre-emergence application of dicamba, with less than 10% injury rates at even 9x the labeled application rate (5,040 g/ha, 4.5 lb/acre; Table 1). The inventors found that, even using an 18x application rate of 10,080 g/ha (9 lb/acre), injury to transgenic dicamba tolerant plants was less than 20% (Table 4). At an approximately 2x rate of 25 application of 1122 g/ha, less than 2% injury was observed. It was therefore indicated the improved weed control associated with pre- and post-emergence applications of herbicides may be used without any significant decreases in productivity due to herbicide damage. Pre-emergent applications of dicamba according to the invention may therefore be combined with one or more herbicide applications post-emergence 30 to dicamba-tolerant plants, while maintaining crop yield and obtaining improved weed control. For example, one such herbicide application regime involved a late pre-emergence application of dicamba in conjunction with a post-emergence application of dicamba at the V2 stage of development. In certain embodiments, the

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517 post-emergence application may be carried out at any point from emergence to harvest. Particularly beneficial will be post-emergence application at any V stage until the soybean canopy closes, for example, at about the VI, V2, V3, V4, V5, V6 and/or later stages.

In accordance with the invention, methods and compositions for the control of weeds are provided comprising the use of plants exhibiting tolerance to glyphosate and auxin-like herbicides such as dicamba. As shown in the working examples, dicamba and glyphosate allow use of decreased amounts of herbicide to achieve the same level of control of glyphosate-tolerant weeds and thus this embodiment provides 10 a significant advance for the control of herbicide tolerance in commercial production fields. In one embodiment, a tank mix of glyphosate and dicamba is applied preand/or post-emergence to plants. Glyphosate and dicamba may additionally be applied separately. In order to obtain the ability to use decreased amount of herbicide, the glyphosate and dicamba are preferably applied within a sufficient interval that 15 both herbicides remain active and able to control weed growth.

This embodiment therefore allows use of lower amounts of either herbicide to achieve the same degree of weed control as an application of only one of the herbicides. For example, the invention provides methods of weed control comprising applying in a field planted with transgenic plants having tolerance to dicamba and 20 glyphosate a herbicide composition comprising less than a lx rate of glyphosate and/or dicamba, relative to the standard manufacturer labeled rate. Examples of respective glyphosate and dicamba application rates include from about a 0.5x-0.95x of either herbicide, specifically including about 0.5x, Ο.όχ, 0.7x, 0.8x. 0.85x, 0.9x, and 0.95x of either herbicide and all derivable combinations thereof, as well as higher 25 rates such as 0.97x and 0.99x. Alternatively, in the case of more difficult to control weeds or where a greater degree of weed control is desired, lx and higher application rates may be made in view of the finding herein that even high application rates of dicamba did not significantly damage plants. The lx application rates are set by the manufacturer of a commercially available herbicide formulation and are known to 30 those of skill in the art. For example, the label for Fallow Master™, a glyphosate and dicamba mixture having a ratio of glyphosate:dicamba of about 2:1 recommends application rates of about 451 g/ha (311 ae g/ha glyphosate: 140 ae g/ha dicamba) to

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517

621 ae g/ha (428 ae g/ha glyphosate: 193 ae g/ha dicamba) depending upon the weed species and weed height.

“Glyphosate” refers to N-phosphonomethylglycine and salts thereof. Glyphosate is commercially available in numerous formulations. Examples of these formulations of glyphosate include, without limitation, those sold by Monsanto Company as ROUNDUP®, ROUNDUP® ULTRA, ROUNDUP® ULTRAMAX, ROUNDUP® CT, ROUNDUP® EXTRA, ROUNDUP® BIACTIVE, ROUNDUP® BIOFORCE, RODEO®, POLARIS®, SPARK® and ACCORD® herbicides, all of which contain glyphosate as its isopropylammonium salt, ROUNDUP®

WEATHERMAX containing glyphosate as its potassium salt; ROUNDUP® DRY and RIVAL® herbicides, which contain glyphosate as its ammonium salt; ROUNDUP® GEOFORCE, which contains glyphosate as its sodium salt; and TOUCHDOWN® herbicide, which contains glyphosate as its trimethylsulfonium salt. “Dicamba” refers to 3,6-dichloro-o-anisic acid or 3,6-dichloro-2-methoxy benzoic acid and its acids and salts. Its salts include isopropylamine, diglycoamine, dimethylamine, potassium and sodium. Examples of commercial formulations of dicamba include, without limitation, Banvel™ (as DMA salt), Clarity™ (as DGA salt), VEL-58-CS-11™ and Vanquish™ (as DGA salt, BASF).

Non-limiting examples of weeds that can be effectively controlled using dicamba are the following: cheese weed, chick weed, while clover, cocklebur, Asiatic dayflower, deadnettle, red stem filaree, Carolina geranium, hemp sesbania, henbit, field horsetail (marestail), knotweed, kochia, lambsquarter, momingglory, indian mustard, wild mustard, redroot pigweed, smooth pigweed, prickly sida, cutleaf evening primrose, common purslane, common ragweed, gaint ragweed, russian thistle, shepardspurse, Pennsylvania smartweed, spurge, velvetleaf, field violet, wild buckwheat, wild radish, soybeanpurslane, sicklepod, mominglory, wild buckwheat, common ragweed, horseweed (marestail), hairy fleabane, buckhom plantain, and palmer pigweed. Non-limiting examples of weeds that can be controlled using dicamba and glyphosate are the following: bamyardgrass, downy brome, volunteer cereals, Persian darnel, field sandbur, green foxtail, wild oats, wild buckwheat, volunteer canola, cowcockle, flixweed, kochia, ladysthumb, lambsquarters, wild mustard, prickly lettuce, redroot pigweed, smartweed, stinkgrass, stinkweed, russian thistle, foxtail, and witchgrass. Combining glyphosate and dicamba achieves the

CA 02653739 2013-05-10 same level of weed control with reduced herbicide amounts and thus the spectrum of weeds that may be controlled at any given herbicide application rate may be increased when the herbicides are combined.

Transgenic plants having herbicide tolerance may be made as described in the art. Dicamba tolerance may be conferred, for example, by a gene for dicamba monooxygenase (DM0) from Pseudomonas maltophilia (US Patent Application No: 20030135879). Examples of sequences that may be used in this regard are nucleic acid encoding the polypeptides of SEQ ID Nos: 2, 4, 6, 8, 10, and 12. Examples of sequences encoding these polypeptides are given as SEQ ID NOS: 1, 3, 5, 7, 9, and

11. SEQ ID NO: 1 shows DM0 from Pseudomonas maltophilia optimized for expression in dicots using Arabidopsis thaliana codon usage. The polypeptide, predicted to have an Ala, Thr, Cys at positions 2, 3, 112, respectively, is given in SEQ ID NO:2. SEQ ID NO:3 shows another Pseudomonas maltophilia DM0 optimized for expression in dicots and encoding the polypeptide of SEQ ID NO:4, predicted to have an Leu, Thr, Cys at positions 2, 3, 112, respectively. SEQ ID NO:5 shows the coding sequence and SEQ ID NO:6 the polypeptide for dicot optimized DM0 predicted to have a Leu, Thr, Trp at positions 2, 3, 112, respectively. SEQ ID NOS:7 and 8 show the coding and polypeptide sequences for DM0 predicted to have an Ala, Thr, Cys at position 2, 3, 112, respectively. SEQ ID NOS:9 and 10 show the dicotoptimized coding sequence and polypeptide sequences for DM0 predicted to have an Ala, Thr, Trp at positions 2, 3, 112, respectively. SEQ ID NOS:11 and 12 show coding sequence and polypeptide sequences for DM0 from Pseudomonas maltophilia (US Patent Application No: 20030135879). Another exemplary DM0 sequence may be a DMO predicted to have a Leu, Thr, Cys at position 2, 3, 112, respectively with codon usage of Pseudomonas maltophilia (US Patent Application No: 20030135879).

Sequences conferring glyphosate tolerance are also known, including glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthases (EPSPS) as described in U.S. Patent 5,627,061, U.S. Patent 5,633,435, U.S. Patent 6,040,497, U.S. Patent 5,094,945; WO04074443; W004009761; by expression of nucleic acids encoding glyphosate degrading enzymes, for example, glyphosate oxidoreductase (GOX, U.S. Patent 5,463,175), glyphosate decarboxylase (W005003362; U.S. Patent Application 20040177399);

CA 02653739 2013-05-10 and by expression of nucleic acids encoding glyphosate inactivating enzymes, such as glyphosate-N-acetyl transferase (GAT, e.g. U.S. Patent publications 20030083480 and 20070079393).

Variants of proteins having a capability to degrade auxin-like herbicides, glyphosate or other herbicides can readily be prepared and assayed for activity according to standard methods. Such sequences can also be identified by techniques know in the art, for example, from suitable organisms including bacteria that degrade auxin-like herbicides such as dicamba or other herbicides (U.S. Patent 5,445,962; Cork and Krueger, 1991; Cork and Khalil, 1995). One means of isolating a DM0 or other sequence is by nucleic acid hybridization, for example, to a library constructed from the source organism, or by RT-PCR using mRNA from the source organism and primers based on the disclosed desaturases. The invention therefore encompasses use of nucleic acids hybridizing under stringent conditions to a DM0 encoding sequence described herein. One of skill in the art understands that conditions may be rendered less stringent by increasing salt concentration and decreasing temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results. An example of high stringency conditions is 5X SSC, 50% formamide and 42°C. By conducting a wash under such conditions, for example, for 10 minutes, those sequences not hybridizing to a particular target sequence under these conditions can be removed.

Variants can also be chemically synthesized, for example, using the known DM0 polynucleotide sequences according to techniques well known in the art. For instance, DNA sequences may be synthesized by phosphoamidite chemistry in an automated DNA synthesizer. Chemical synthesis has a number of advantages. In particular, chemical synthesis is desirable because codons preferred by the host in which the DNA sequence will be expressed may be used to optimize expression. Not all of the codons need to be altered to obtain improved expression, but preferably at least the codons rarely used in the host are changed to host-preferred codons. High levels of expression can be obtained by changing greater than about 50%, most preferably at least about 80%, of the codons to host-preferred codons. The codon preferences of many host cells are known (PCT WO 97/31115; PCT WO 97/11086; EP 646643; EP 553494; and U.S. Patent Nos: 5,689,052; 5,567,862; 5,567,600; 5,552,299 and 5,017,692). The codon preferences of other host cells can be deduced

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517 by methods known in the art. Also, using chemical synthesis, the sequence of the DNA molecule or its encoded protein can be readily changed to, for example, optimize expression (for example, eliminate mRNA secondary structures that interfere with transcription or translation), add unique restriction sites at convenient points, and 5 delete protease cleavage sites.

Modification and changes may be made to the polypeptide sequence of a protein such as the DM0 sequences provided herein while retaining enzymatic activity. The following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, modified polypeptide and 10 corresponding coding sequences. It is known, for example, that certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein’s biological functional activity, certain amino acid sequence substitutions can 15 be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the DM0 peptide sequences described herein or other herbicide tolerance polypeptides and corresponding DNA coding sequences without appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte et al., 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines 25 the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte et al., 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine 30 (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517

It is known in the art that amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are 5 within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101 states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of 10 its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 + 1); glutamate (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 + 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0);

methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are 20 particularly preferred, and those within ±0.5 are even more particularly preferred.

Exemplary substitutions which take these and various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

A gene conferring herbicide tolerance will typically be linked to a plant promoter driving expression of the gene in an amount sufficient to confer the herbicide tolerance. Promoters suitable for this and other uses are well known in the art. Examples describing such promoters include U.S. Patent 6,437,217 (maize RS81 promoter), U.S. Patent 5,641,876 (rice actin promoter), U.S. Patent 6,426,446 (maize 30 RS324 promoter), U.S. Patent 6,429,362 (maize PR-1 promoter), U.S. Patent

6,232,526 (maize A3 promoter), U.S. Patent 6,177,611 (constitutive maize promoters), U.S. Patents 5,322,938, 5,352,605, 5,359,142 and 5,530,196 (35S promoter), U.S. Patent 6,433,252 (maize L3 oleosin promoter), U.S. Patent 6,429,357

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517 (rice actin 2 promoter as well as a rice actin 2 intron), U.S. Patent 5,837,848 (root specific promoter), U.S. Patent 6,294,714 (light inducible promoters), U.S. Patent 6,140,078 (salt inducible promoters), U.S. Patent 6,252,138 (pathogen inducible promoters), U.S. Patent 6,175,060 (phosphorus deficiency inducible promoters), U.S.

Patent 6,388,170 (PC1SV promoter), U.S. Patent 6,635,806 (gamma-coixin promoter), and U.S. patent application Serial No. 09/757,089 (maize chloroplast aldolase promoter). Additional promoters that may find use are a nopaline synthase (NOS) promoter (Ebert et al., 1987), the octopine synthase (OCS) promoter (which is carried on tumor-inducing plasmids of Agrobacterium tumefaciens), the caulimovirus 10 promoters such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., 1987), the CaMV 35S promoter (Odell et al., 1985), the figwort mosaic virus 35Spromoter (Walker et al., 1987), the sucrose synthase promoter (Yang et al., 1990), the R gene complex promoter (Chandler et al., 1989), the chlorophyll a/b binding protein gene promoter, CaMV35S (U.S. Patent Nos. 5,322,938; 5,352,605; 5,359,142; and 15 5,530,196), FMV35S (U.S. Patents 6,051,753; 5,378,619), a PC1SV promoter (US

Patent 5,850,019; or SEQ ID NO:20), the SCP promoter (U.S. Pat. No. 6,677,503), and AGRtu.nos (GenBank Accession V00087; Depicker et al, 1982; Bevan et al., 1983) promoters, and the like (see also see Table 1).

Benefit may be obtained for the expression of herbicide tolerance genes by use 20 of a sequence coding for a transit peptide. For example, incorporation of a suitable chloroplast transit peptide, such as, the Arabidopsis thaliana EPSPS CTP (Klee et al., 1987), and the Petunia hybrida EPSPS CTP (della-Cioppa et al., 1986) has been shown to target heterologous EPSPS protein sequences to chloroplasts in transgenic plants. Chloroplast transit peptides (CTPs) are engineered to be fused to the N25 terminus of a protein to direct the protein into the plant chloroplast. Such sequences may find use in connection with a nucleic acid conferring dicamba tolerance in particular. Many chloroplast-localized proteins are expressed from nuclear genes as precursors and are targeted to the chloroplast by a chloroplast transit peptide that is removed during the import process. Examples of chloroplast proteins include the 30 small subunit (RbcS2) of ribulose-1,5,-bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, the light-harvesting complex protein I and protein II, and thioredoxin F. Other exemplary chloroplast targeting sequences include the maize cab-m7 signal sequence (Becker et al., 1992; PCT WO 97/41228), the pea glutathione

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517 reductase signal sequence (Creissen et al., 1995; PCT WO 97/41228), and the CTP of the Nicotiana tabacum ribulose 1,5-bisphosphate carboxylase small subunit chloroplast transit peptide (SSU-CTP) (Mazur, et al., 1985). Use of AtRbcS4 (CTP1;

U.S. Patent 5,728,925), AtShkG (CTP2; Klee et al., 1987), AtShkGZm (CTP2synthetic; see SEQ ID NO:14 of W004009761), and PsRbcS (Coruzzi et al., 1984), as well as others disclosed, for instance, in U.S. Provisional Patent Application 60/891,675, peptide and nucleic acid sequences for which are listed herein at SEQ ID NOs:21-32, may be of benefit for use with the invention.

A 5’ UTR that functions as a translation leader sequence is a DNA genetic 10 element located between the promoter sequence of a gene and the coding sequence.

The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences include maize and petunia heat shock 15 protein leaders (U.S. Patent No. 5,362,865), plant virus coat protein leaders, plant rubisco leaders, among others (Turner and Foster, 1995). Non-limiting examples of 5’ UTRs that may in particular be of benefit for use GmHsp (U.S. Patent 5,659,122), PhDnaK (U.S. Patent 5,362,865), AtAntl, TEV (Carrington and Freed, 1990), and AGRtunos (GenBank Accession V00087; Bevan et al., 1983).

The 3’ non-translated sequence, 3’ transcription termination region, or poly adenylation region means a DNA molecule linked to and located downstream of a structural polynucleotide molecule and includes polynucleotides that provide polyadenylation signal and other regulatory signals capable of affecting transcription, mRNA processing or gene expression. The polyadenylation signal functions in plants 25 to cause the addition of polyadenylate nucleotides to the 3’ end of the mRNA precursor. The polyadenylation sequence can be derived from the natural gene, from a variety of plant genes, or from T-DNA genes. An example of a 3’ transcription termination region is the nopaline synthase 3’ region (nos 3’; Fraley et al., 1983). The use of different 3’ nontranslated regions is exemplified (Ingelbrecht et al., 1989).

Polyadenylation molecules from a Pisum sativum RbcS2 gene (Ps.RbcS2-E9; Coruzzi et al., 1984) and AGRtu.nos (Rojiyaa et al., 1987, Genbank Accession E0f3f2) in particular may be of benefit for use with the invention.

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517

Intron sequences are known in the art to aid in the expression of transgenes in monocot plant cells. Examples of introns include the com actin intron (US Patent 5,641,876), the com HSP70 intron (ZmHSP70; US Patent 5,859,347; US Patent 5,424,412), and rice TPI intron (OsTPI; US Patent No. 7,132,528), and are of benefit 5 in practicing this invention.

Any of the techniques known in the art for introduction of transgenes into plants may be used to prepare a herbicide tolerant plant in accordance with the invention (see, for example, Miki et al., 1993). Suitable methods for transformation of plants are believed to include virtually any method by which DNA can be 10 introduced into a cell, such as by electroporation as illustrated in U.S. Patent No. 5,384,253; microprojectile bombardment as illustrated in U.S. Patent Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,403,865; Agrobacterium-mediated transformation as illustrated in U.S. Patent Nos. 5,635,055; 5,824,877; 5,591,616; 5,981,840; and 6,384,301; and protoplast transformation as illustrated in U.S. Patent 15 No. 5,508,184, etc. Through the application of techniques such as these, the cells of virtually any plant species may be stably transformed, and these cells developed into transgenic plants. Techniques that may be particularly useful in the context of cotton transformation are disclosed in U.S. Patent Nos. 5,846.797, 5,159,135, 5,004,863, and 6,624,344; and techniques for transforming Brassica plants in particular are disclosed, 20 for example, in U.S. Patent 5,750,871; and techniques for transforming soybean are disclosed in for example in Zhang et al., 1999 and US Patent 6,384,301). Com can be transformed using methods described in WO9506722 and US patent application 20040244075.

After effecting delivery of exogenous DNA to recipient cells, the next steps 25 generally concern identifying the transformed cells for further culturing and plant regeneration. In order to improve the ability to identify transformants, one may desire to employ a selectable or screenable marker gene with a transformation vector prepared in accordance with the invention. In this case, one would then generally assay the potentially transformed cell population by exposing the cells to a selective 30 agent or agents, or one would screen the cells for the desired marker gene trait.

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, any suitable plant tissue culture

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517 media, for example, MS and N6 media may be modified by including further substances such as growth regulators. 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 5 tissue is suitable for regeneration, typically at least 2 weeks, then transferred to media conducive to shoot formation. Cultures are transferred periodically until sufficient shoot formation has occurred. Once shoot are formed, they are transferred to media conducive to root formation. Once sufficient roots are formed, plants can be transferred to soil for further growth and maturity.

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.

Once a transgene has been introduced into a plant, that gene can be introduced into any plant sexually compatible with the first plant by crossing, without the need for ever directly transforming the second plant. Therefore, as used herein the term 20 “progeny” denotes the offspring of any generation of a parent plant prepared in accordance with the instant invention, wherein the progeny comprises a selected DNA construct prepared in accordance with the invention. A “transgenic plant” may thus be of any generation. “Crossing” a plant to provide a plant line having one or more added transgenes or alleles relative to a starting plant line, as disclosed herein, is 25 defined as the techniques that result in a particular sequence being introduced into a plant line by crossing a starting line with a donor plant line that comprises a transgene or allele of the invention. To achieve this one could, for example, perform the following steps: (a) plant seeds of the first (starting line) and second (donor plant line that comprises a desired transgene or allele) parent plants; (b) grow the seeds of the 30 first and second parent plants into plants that bear flowers; (c) pollinate a flower from the first parent plant with pollen from the second parent plant; and (d) harvest seeds produced on the parent plant bearing the fertilized flower.

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517

The preparation of herbicide compositions for use in connection with the current invention will be apparent to those of skill in the art in view of the disclosure. Such compositions, which are commercially available, will typically include, in addition to the active ingredient, components such as surfactants, solid or liquid 5 carriers, solvents and binders. Examples of surfactants that may be used for application to plants include the alkali metal, alkaline earth metal or ammonium salts of aromatic sulfonic acids, e.g., ligno-, phenol-, naphthalene- and dibutylnaphthalenesulfonic acid, and of fatty acids of arylsulfonates, of alkyl ethers, of lauryl ethers, of fatty alcohol sulfates and of fatty alcohol glycol ether sulfates, 10 condensates of sulfonated naphthalene and its derivatives with formaldehyde, condensates of naphthalene or of the naphthalenesulfonic acids with phenol and formaldehyde, condensates of phenol or phenolsulfonic acid with formaldehyde, condensates of phenol with formaldehyde and sodium sulfite, polyoxyethylene octylphenyl ether, ethoxylated isooctyl-, octyl-or nonylphenol, tributylphenyl 15 polyglycol ether, alkylaryl polyether alcohols, isotridecyl alcohol, ethoxylated castor oil, ethoxylated triarylphenols, salts of phosphated triarylphenolethoxylates, lauryl alcohol polyglycol ether acetate, sorbitol esters, lignin-sulfite waste liquors or methylcellulose, or mixtures of these. Common practice in the case of surfactant use is about 0.25% to 1.0% by weight, and more commonly about 0.25% to 0.5% by 20 weight.

Compositions for application to plants may be solid or liquid. Where solid compositions are used, it may be desired to include one or more carrier materials with the active compound. Examples of carriers include mineral earths such as silicas, silica gels, silicates, talc, kaolin, attaclay, limestone, chalk, loess, clay, dolomite, 25 diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea and urea, products of vegetable origin such as cereal meals, tree bark meal, wood meal and nutshell meal, cellulose powders, attapulgites, montmorillonites, mica, vermiculites, synthetic silicas and synthetic calcium silicates, 30 or mixtures of these.

For liquid solutions, water-soluble compounds or salts may be included, such as sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, sodium acetate, ammonium hydrogen sulfate, ammonium chloride, ammonium acetate,

CA 02653739 2013-05-10 ammonium formate, ammonium oxalate, ammonium carbonate, ammonium hydrogen carbonate, ammonium thiosulfate, ammonium hydrogen diphosphate, ammonium dihydrogen monophosphate, ammonium sodium hydrogen phosphate, ammonium thiocyanate, ammonium sulfamate or ammonium carbamate.

Other exemplary components in herbicidal compositions include binders such as polyvinylpyrrolidone, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, carboxymethylcellulose, starch, vinylpyrrolidone/vinyl acetate copolymers and polyvinyl acetate, or mixtures of these; lubricants such as magnesium stearate, sodium stearate, talc or polyethylene glycol, or mixtures of these; antifoams such as silicone emulsions, long-chain alcohols, phosphoric esters, acetylene diols, fatty acids or organofluorine compounds, and complexing agents such as: salts of ethylenediaminetetraacetic acid (EDTA), salts of trinitrilotriacetic acid or salts of polyphosphoric acids, or mixtures of these.

Equipment and methods known in the art are used to apply various herbicide treatments as disclosed herein. The application rates of herbicides maybe varied, for instance as described above, depending upon the soil texture, pH, organic matter content, tillage systems, and the size of the weed, and can be determined by consulting the herbicide label for the proper herbicide rate.

EXAMPLES

The following examples are included to illustrate embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention. 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, 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.

CA 02653739 2013-05-10

Example 1

Tolerance of soybean plants containing DMO-encoding polynucleotide construct to early pre-emergence application of dicamba

Transgenic soybean plants were obtained by Agrobacterium transformation of soybean cotyledonary nodes using standard procedures and a binary vector containing the DMO-encoding polynucleotide given as SEQ ID NO:7, which encodes the polypeptide of SEQ ID NO:8. Four transgenic soybean events were prepared and designated Events 1-4. Transgenic soybean plants containing the events were tested for their tolerance to dicamba herbicide relative to controls, confirming herbicide tolerance. Non-transgenic soybean plants were used as controls.

Transgenic and control soybean seeds were planted into 3.5-inch square plastic pots containing Redi-earth™ (Scotts-Sierra Horticultural Products Co., Marysville, Ohio). The soil surface was treated with various amounts (561 to 5040 g/ha, 0.5 to 4.5 lb/acre, or lx to 9x labeled rates) of dicamba formulations (Clarity™ or Banvel™, BASF, Raleigh, NC). The pots were placed on capillary matting in 35 inch x 60 inch fiberglass watering trays for overhead and/or sub-irrigation for the duration of the test period so as to maintain optimum soil moisture for plant growth and were fertilized with Osmocote™ (14-14-14 slow release; Scotts-Sierra Horticultural Products Co., Marysville, Ohio) at the rate of 100 gm/crnft. to sustain plant growth for the duration of greenhouse trials.

The plants were grown in greenhouses at 27°/21 °C day/night temperature with relative humidity between 25%-75% to simulate warm season growing conditions of late spring. A 14 h minimum photoperiod was provided with supplemental light at about 600 μΕ as needed. Trials were established in a randomized block design randomized by rate with 4 to 6 replications of each treatment depending on plant quality, availability, and to account for any environmental variability that may have occurred within the confines of each greenhouse.

Treated plants in greenhouse trials were visually assessed at a particular day after treatment (DAT) for injury on a scale of 0 to 100 percent relative to untreated control plants, with zero representing “no” injury and 100% representing “complete” injury or death. Data were collected and analyzed using suitable statistical methods.

CA 02653739 2013-05-10

The results of the study surprisingly showed that soybean plants transformed with the DMO-en coding polynucleotide construct were tolerant to even early preemergence application of dicamba. As indicated in Table 1 below, injury to the transgenic plants was less than 10% even at the highest application rate z.e., 5040 g/ha, 4.5 lb/acre, or 9x labeled rates of dicamba.

Table 1. Percentage injury to non-transgenic or transgenic soybean plants from early pre-emergence application of dicamba at sowing. The % injury was represented as ANOVA mean comparisons. Similar letters represent no statistical difference at the p=0.05 level.

<td> Formulation</td><td> ID</td><td colspan="9"> % injury at shown rates (g ae/ha*) at 14 DAT</td><td></td>

<td> Clarity™</td><td></td><td> 561</td><td></td><td> 840</td><td></td><td> 2244</td><td></td><td> 4485</td><td></td><td> 5040</td><td></td>

<td></td><td> Control</td><td> 67.0</td><td> a</td><td> 73.0</td><td> b</td><td> 96.6</td><td> a</td><td> 98.2</td><td> a</td><td> 99.5</td><td> a</td>

<td></td><td> Control</td><td> 61.0</td><td> a</td><td> 86.0</td><td> a</td><td> 98.1</td><td> a</td><td> 98.3</td><td> a</td><td> 99.8</td><td> a</td>

<td></td><td> Event 1</td><td> 0.0</td><td> b</td><td> 0.0</td><td> c</td><td> 1.7</td><td> be</td><td> 0.7</td><td> b</td><td> 3.1</td><td> b</td>

<td></td><td> Event. 2</td><td> 0.0</td><td> b</td><td> 0.0</td><td> c</td><td> 1.1</td><td> c</td><td> 1.0</td><td> b</td><td> 2.2</td><td> b</td>

<td></td><td> Event 3</td><td> 0.0</td><td> b</td><td> 0.0</td><td> c</td><td> 1.1</td><td> c</td><td> 0.6</td><td> b</td><td> 3.5</td><td>^b</td>

<td></td><td> Event 4</td><td> 0.0</td><td> b</td><td> 0.0</td><td> c</td><td> 4.4</td><td> b</td><td> 0.8</td><td> b</td><td> 7.2</td><td> b</td>

<td></td><td> LSD</td><td> 9.9</td><td></td><td> 7.2</td><td></td><td> 3.2</td><td></td><td> 2.2</td><td></td><td> 5.1</td><td></td>

Example 2

Tolerance of soybean plants containing a DMO-encoding polynucleotide construct to early pre-emergence application of dicamba at sowing followed by post-emergence application of dicamba in addition to the method described in Example 2 for early pre-emergence (at sowing) application of dicamba, post-emergence ÇV2 stage of soybean development) application of dicamba was made with a track sprayer using the Teejet™ 950IE flat fan nozzle (Spraying Systems Co, Wheaton, IL) with the air pressure set at a minimum of 24 psi (165kpa). The spray nozzle was kept at a height of about 16 inches above the

CA 02653739 2008-11-28

WO 2007/143690

PCT/US2007/070517 top of the plant matenal for spraying. The spray volume was 10 gallons per acre or 93 liters per hectare.

As shown in Table 2, soybean plants transformed with the DMO-encoding polynucleotide construct were tolerant to early pre-emergence applications of dicamba at sowing followed by post-emergence application of dicamba. Surprisingly, injury to transgenic plants was less than 20% at the overall dicamba rate of 10080 g/ha, 9 lb/acre or 18x labeled rate.

Table 2. Percentage injury to non-transgenic or transgenic soybean plants from application of dicamba at sowing followed by post-emergence application at V2 stage.*

<td> Formulation</td><td> Plants</td><td colspan="2"> % in.</td><td colspan="8"> ury at shown rates (g ae/ha*) at 28 DAT</td>

<td> Clarity™</td><td></td><td> 1122</td><td></td><td> 1680</td><td></td><td> 4488</td><td></td><td> 8970</td><td></td><td> 10080</td><td></td>

<td></td><td> Control</td><td> 97.5</td><td> a</td><td> 98.8</td><td> a</td><td> 99.8</td><td> a</td><td> 100.0</td><td> a</td><td> 100.0</td><td> a</td>

<td></td><td> Control</td><td> 95.6</td><td> a</td><td> 98.1</td><td> a</td><td> 99.4</td><td> a</td><td> 100.0</td><td> a</td><td> 100.0</td><td> a</td>

<td></td><td> Event 1</td><td> 0.0</td><td> c</td><td> 1.8</td><td> b</td><td> 4.5</td><td> d</td><td> 11.9</td><td> c</td><td> 16.9</td><td> b</td>

<td></td><td> Event 2</td><td> 2.6</td><td> be</td><td> 3.9</td><td> b</td><td> 8.1</td><td> be</td><td> 13.8</td><td> b</td><td> 16.9</td><td> b</td>

<td></td><td> Event 3</td><td> 3.1</td><td> b</td><td> 2.9</td><td> b</td><td> 8.8</td><td> b</td><td> 11.9</td><td> c</td><td> 17.5</td><td> b</td>

<td></td><td> Event 4</td><td> 2.3</td><td> be</td><td> 2.0</td><td> b</td><td> 6.9</td><td> c</td><td> 11.9</td><td> c</td><td> 15.6</td><td> b</td>

<td></td><td> LSD</td><td> 3.1</td><td></td><td> 2.2</td><td></td><td> 1.4</td><td></td><td> 1.6</td><td></td><td> 1.9</td><td></td>

*The % injury was represented as ANOVA mean comparisons. Similar letters represent no statistical difference at the p=0.05 level.

Example 3

Tolerance of soybean plants containing DMO-encoding polynucleotide construct to late pre-emergence application of dicamba

An analysis was carried out of the effect of late pre-emergence applications of dicamba at soil cracking due to emergence of soybean seedling hypocotyls. Dicamba applications were made using a track sprayer as described in the previous examples. As shown in Table 3, soybean plants transformed with the DMO-encoding polynucleotide construct were found to be tolerant to late pre-emergence application of dicamba at soil cracking. Significantly, injury in the transgenic events was less than 5% even at the highest rate i.e., 5040 g/ha, 4.5 lb/acre, or 9x labeled rates of dicamba.

CA 02653739 2008-11-28

WO 2007/143690

PCT/US2007/070517

Table 3. Percentage injury to non-transgenic or transgenic soybean plants from late pre-emergence application of dicamba at soil cracking.*

<td> Formulation</td><td> Plants</td><td colspan="10"> % injury at shown rates (g ae/ha*) at 14 DAT</td>

<td> Clarity™</td><td></td><td> 561</td><td></td><td> 840</td><td></td><td> 2244</td><td></td><td> 4485</td><td></td><td> 5040</td><td></td>

<td></td><td> Control</td><td> 86.9</td><td> a</td><td> 96.8</td><td> a</td><td> 98.4</td><td> A</td><td> 98.5</td><td> a</td><td> 99.2</td><td> a</td>

<td></td><td> Control</td><td> 89.6</td><td> a</td><td> 91.9</td><td> a</td><td> 98.4</td><td> A</td><td> 99.0</td><td> a</td><td> 99.4</td><td> a</td>

<td></td><td> Event 1</td><td> 0.0</td><td> b</td><td> 0.0</td><td> b</td><td> 0.5</td><td> C</td><td> 2.5</td><td> be</td><td> 2.0</td><td> b</td>

<td></td><td> Event 2</td><td> 0.0</td><td> b</td><td> 0.0</td><td> b</td><td> 2.9</td><td> be</td><td> 0.0</td><td> c</td><td> 1.5</td><td> b</td>

<td></td><td> Event 3</td><td> 0.0</td><td> b</td><td> 0.0</td><td> b</td><td> 1.5</td><td> be</td><td> 4.4</td><td> b</td><td> 1.3</td><td> b</td>

<td></td><td> Event 4</td><td> 0.0</td><td> b</td><td> 0.5</td><td> b</td><td> 3.3</td><td> B</td><td> 3.0</td><td> be</td><td> 1.3</td><td> b</td>

<td></td><td> LSD</td><td> 8.1</td><td></td><td> 5.4</td><td></td><td> 2.4</td><td></td><td> 3.9</td><td></td><td> 2.3</td><td></td>

* The % injury was represented as ANOVA mean comparisons. Similar letters represent no statistical difference at the p=0.05 level.

Example 4

Tolerance of soybean plants containing DMO-encoding polynucleotide construct to late pre-emergence applications of dicamba followed by post-emergence applications of dicamba

In addition to the studies above, an analysis was carried out of the effect of late pre-emergence applications of dicamba at soil cracking followed by postemergence application of dicamba at the V2 stage of development. As shown in Table 4, soybean plants transformed with the DMO-encoding polynucleotide construct were tolerant to late pre-emergence application of dicamba at soil cracking and post-emergence application of dicamba. Injury to transgenic events was less than 20% even at the overall dicamba rate of 10080 g/ha, 9 lb/acre, or 18x labeled rate.

CA 02653739 2008-11-28

WO 2007/143690

PCT/US2007/070517

Table 4. Percentage injury to non-transgenic or transgenic soybean plants from late pre-emergence application of dicamba at soil cracking followed by postemergence application at V2 stage.*

<td> Formulation</td><td> Plants</td><td colspan="10"> % injury at shown rates (g ae/ha*) at 28 DAT</td>

<td> Clarity™</td><td></td><td> 1122</td><td></td><td> 1680</td><td></td><td> 4488</td><td></td><td> 8970</td><td></td><td> 10080</td><td></td>

<td></td><td> Control</td><td> 95.6</td><td> a</td><td> 98.1</td><td> a</td><td> 100.0</td><td> a</td><td> 100.0</td><td> a</td><td> 100.0</td><td> a</td>

<td></td><td> Control</td><td> 95.0</td><td> a</td><td> 98.1</td><td> a</td><td> 99.4</td><td> a</td><td> 99.8</td><td> a</td><td> 100.0</td><td> a</td>

<td></td><td> Event 1</td><td> 0.3</td><td> b</td><td> 0.9</td><td> b</td><td> 6.3</td><td> b</td><td> 13.1</td><td> b</td><td> 16.3</td><td> be</td>

<td></td><td> Event 2</td><td> 0.8</td><td> b</td><td> 1.6</td><td> b</td><td> 6.0</td><td> b</td><td> 11.3</td><td> c</td><td> 15.0</td><td> c</td>

<td></td><td> Event 3</td><td> 1.0</td><td> b</td><td> 1.4</td><td> b</td><td> 7.5</td><td> b</td><td> 11.3</td><td> c</td><td> 17.5</td><td> b</td>

<td></td><td> Event 4</td><td> 1.8</td><td> b</td><td> 1.8</td><td> b</td><td> 7.5</td><td> b</td><td> 13.1</td><td> b</td><td> 16.3</td><td> be</td>

<td></td><td> LSD</td><td> 4.5</td><td></td><td> 2.7</td><td></td><td> 1.6</td><td></td><td> 1.6</td><td></td><td> 1.9</td><td></td>

* The % injury was represented as ANOVA mean comparisons. Similar letters 5 represent no statistical difference at the p=0.05 level.

Example 5

Tolerance of soybean plants containing DMO-encoding polynucleotide construct to pre- and post-emergence application of dicamba in the field

Non-transgenic and transgenic soybean seeds were planted around the beginning of the growing season at the time of optimum growth conditions depending on soil moisture, temperature, and seeding depth. Across all locations seeds were planted under split-plot design with dicamba treatments as whole-plot effects and events as split-plot effects. The design details were as follows: 6 locations, 2 replications/location, 2 rows/plot, row length 12 feet (+3 ft alley), 9 seeds/foot, 108 seeds/row, 5 events (Events 1-4 and a fifth event that was segregating); and 4 treatments as shown below in Table 5. In all 240 plots were planted at 6 locations (40 per location).

CA 02653739 2008-11-28

WO 2007/143690

PCT/US2007/070517

Table 5. Details of 4 treatments applied to show the tolerance of transgenic soybean to dicamba.

<td></td><td colspan="2"> 1 st Application</td><td colspan="2"> 2nd Application</td>

<td> Treatment</td><td> Rate</td><td> Plant Stage</td><td> Rate</td><td> Plant Stage</td>

<td> 1</td><td> NO Dicamba</td><td> NO Dicamba</td><td> NO Dicamba</td><td> NO Dicamba</td>

<td> 2</td><td> 1.5 lb ae/acre</td><td> At Planting</td><td> N/A</td><td> N/A</td>

<td> 3</td><td> N/A</td><td> N/A</td><td> 1.5 lb ae/acre</td><td> V3-4</td>

<td> 4</td><td> 1.5 lb ae/acre</td><td> At Planting</td><td> 1.5 lb ae/acre</td><td> V3-4</td>

Four non-transgenic border rows were planted all around the trial using a known commercial line such as A3525. Optimum production and management practices known in the art were followed. Maximum pest control and disease control was practiced as needed to prevent confounding effects of dicamba applications. The field was irrigated as needed according to standard practices.

All plants in the field were treated with pre-emergence and post-emergence 10 applications of dicamba and visually assessed at a particular day after planting for injury on a scale of 0 to 100 percent relative to untreated control plants, with zero representing “no” injury and 100% representing “complete” injury or death. Seed planting and pre-emergence treatment were carried out approximately one-month apart in late spring in Monmouth, IL. As shown in Table 6, it was found that all 15 transgenic soybean plants had no or very little injury. A fifth transgenic event used appeared to be segregating, so a certain percentage of plants died after the treatments.

Table 6. Tolerance of soybean plants containing DMO-encoding polynucleotide construct to pre- and post-emergence application of dicamba in field.*

<td> Event #</td><td> Trmt</td><td> % Ini</td><td> % In.i</td><td> % In.i</td><td> % In.i</td><td> % GR</td><td> % Ini</td><td> % GR</td><td rowspan="2"> Dead or Stunted</td>

<td></td><td></td><td> 6/7</td><td> 6/13</td><td> 6/20</td><td> 6/27</td><td> 6/27</td><td> 7/5</td><td> 7/5</td>

<td> 1</td><td> No spray</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 2</td><td> 0</td><td> 0</td>

<td> 1</td><td> No spray</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 2</td><td> No spray</td><td> 0</td><td> 0</td><td> 0</td><td> 1</td><td> 0</td><td> 3</td><td> 3</td><td> 0</td>

<td> 2</td><td> No spray</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 3</td><td> No spray</td><td> 0</td><td> 0</td><td> 0</td><td> 1</td><td> 0</td><td> 3</td><td> 0</td><td> 0</td>

<td> 3</td><td> No spray</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 3</td><td> 0</td><td> 0</td>

<td> 4</td><td> No spray</td><td> 0</td><td> 0</td><td> 0</td><td> 2</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 4</td><td> No spray</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 5</td><td> 0</td><td> 0</td>

<td> 5</td><td> No spray</td><td> 0</td><td> 0</td><td> 0</td><td> 7</td><td> 0</td><td> 5</td><td> 2</td><td> 0</td>

<td> 5</td><td> No spray</td><td> 0</td><td> 0</td><td> 0</td><td> 7</td><td> 3</td><td> 7</td><td> 3</td><td> 0</td>

<td> 1</td><td> Pre at sowing</td><td> 0</td><td> 0</td><td> 0</td><td> 1</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

CA 02653739 2008-11-28

WO 2007/143690

PCT/US2007/070517

<td> Event #</td><td> Trmt</td><td> % Inj</td><td> % Inj</td><td> % Inj</td><td> % Inj</td><td> % GR</td><td> % Inj</td><td> % GR</td><td rowspan="2"> Dead or Stunted</td>

<td></td><td></td><td> 6/7</td><td> 6/13</td><td> 6/20</td><td> 6/27</td><td> 6/27</td><td> 7/5</td><td> 7/5</td>

<td> 1</td><td> Pre at sowing</td><td> 0</td><td> 2</td><td> 0</td><td> 2</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 2</td><td> Pre at sowing</td><td> 0</td><td> 0</td><td> 0</td><td> 5</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 2</td><td> Pre at sowing</td><td> 0</td><td> 0</td><td> 0</td><td> 2</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 3</td><td> Pre at sowing</td><td> 0</td><td> 4</td><td> 0</td><td> 1</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 3</td><td> Pre at sowing</td><td> 0</td><td> 2</td><td> 0</td><td> 5</td><td> 0</td><td> 3</td><td> 0</td><td> 0</td>

<td> 4</td><td> Pre at sowing</td><td> 0</td><td> 3</td><td> 0</td><td> 5</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 4</td><td> Pre at sowing</td><td> 0</td><td> 4</td><td> 0</td><td> 2</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 5</td><td> Pre at sowing</td><td> 0</td><td> 15</td><td> 15</td><td> 5</td><td> 0</td><td> 0</td><td> 0</td><td> 24</td>

<td> 5</td><td> Pre at sowing</td><td> 0</td><td> 8</td><td> 10</td><td> 2</td><td> 0</td><td> 0</td><td> 0</td><td> 14</td>

<td> 1</td><td> Post at V3</td><td> 0</td><td> 0</td><td> 0</td><td> 5</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 1</td><td> Post at V3</td><td> 0</td><td> 0</td><td> 0</td><td> 7</td><td> 0</td><td> 0</td><td> 0</td><td> 2</td>

<td> 2</td><td> Post at V3</td><td> 0</td><td> 0</td><td> 0</td><td> 3</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 2</td><td> Post at V3</td><td> 0</td><td> 0</td><td> 0</td><td> 3</td><td> 0</td><td> 2</td><td> 0</td><td> 1</td>

<td> 3</td><td> Post at V3</td><td> 0</td><td> 0</td><td> 0</td><td> 3</td><td> 3</td><td> 0</td><td> 0</td><td> 0</td>

<td> 3</td><td> Post at V3</td><td> 0</td><td> 0</td><td> 0</td><td> 5</td><td> 0</td><td> 3</td><td> 0</td><td> 0</td>

<td> 4</td><td> Post at V3</td><td> 0</td><td> 0</td><td> 0</td><td> 3</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 4</td><td> Post at V3</td><td> 0</td><td> 0</td><td> 0</td><td> 3</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 5</td><td> Post at V3</td><td> 0</td><td> 0</td><td> 0</td><td> 7</td><td> 0</td><td> 2</td><td> 0</td><td> 15</td>

<td> 5</td><td> Post at V3</td><td> 0</td><td> 0</td><td> 0</td><td> 5</td><td> 5</td><td> 2</td><td> 0</td><td> 15</td>

<td> 1</td><td> Pre &amp; Post</td><td> 0</td><td> 0</td><td> 0</td><td> 5</td><td> 0</td><td> 0</td><td> 0</td><td> 3</td>

<td> 1</td><td> Pre &amp; Post</td><td> 0</td><td> 2</td><td> 2</td><td> 5</td><td> 3</td><td> 0</td><td> 0</td><td> 0</td>

<td> 2</td><td> Pre &amp; Post</td><td> 0</td><td> 0</td><td> 0</td><td> 1</td><td> 0</td><td> 2</td><td> 0</td><td> 0</td>

<td> 2</td><td> Pre &amp; Post</td><td> 0</td><td> 0</td><td> 0</td><td> 2</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 3</td><td> Pre &amp; Post</td><td> 0</td><td> 0</td><td> 0</td><td> 3</td><td> 0</td><td> 2</td><td> 0</td><td> 0</td>

<td> 3</td><td> Pre &amp; Post</td><td> 0</td><td> 2</td><td> 0</td><td> 3</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 4</td><td> Pre &amp; Post</td><td> 0</td><td> 0</td><td> 0</td><td> 3</td><td> 8</td><td> 2</td><td> 2</td><td> 0</td>

<td> 4</td><td> Pre &amp; Post</td><td> 0</td><td> 1</td><td> 0</td><td> 3</td><td> 3</td><td> 0</td><td> 0</td><td> 0</td>

<td> 5</td><td> Pre &amp; Post</td><td> 0</td><td> 15</td><td> 10</td><td> 3</td><td> 5</td><td> 0</td><td> 0</td><td> 23</td>

<td> 5</td><td> Pre &amp; Post</td><td> 0</td><td> 10</td><td> 10</td><td> 1</td><td> 0</td><td> 0</td><td> 0</td><td> 20</td>

* No spray means no dicamba was applied to the plants. Pre at sowing means 1.5 lb/acre of dicamba was applied at planting. Post at V3 means 1.5 lb/acre of dicamba was applied 4 weeks after planting. Pre and post means 1.5 lb/acre of dicamba was applied at planting and 1.5 lb/acre of dicamba was applied 4 weeks after planting. % inj means percentage injury on given date. % GR means percentage growth reduction.

Example 6

Controlling glyphosate tolerant weeds by dicamba

Marestail is one of the major weeds in a crop field. Marestail is effectively controlled by glyphosate, but the development of methods for controlling this common weed with other herbicides is important to minimize opportunities for herbicide tolerance to develop. An analysis was carried out to determine the extent to which this glyphosate tolerant weed could be controlled by applications of dicamba. Marestail (Conyza canadensis) plants of two biotypes, each from a different

CA 02653739 2008-11-28

WO 2007/143690

PCT/US2007/070517 geographic region, California (CA) and Kentucky (KY), were grown, and treated at 46 inch diameter rosette leaf stage with dicamba as described in Example 2 and 3. The results of the study, as shown in Table 7, demonstrated that dicamba was equally effective in controlling both susceptible and tolerant biotypes of marestail from CA and KY. Dicamba was more effective in controlling resistant biotypes at lower application rates than glyphosate. For example, 2100 g/ha of glyphosate was required to obtain about 77% and 91% inhibition of CA and KY resistant biotypes, whereas only 280 g/ha of dicamba was required to obtain about 83% and about 91% control of CA and KY resistant biotypes.

Table 7. Control of glyphosate tolerant weeds by dicamba.

<td rowspan="2"> Formulation</td><td rowspan="2"> Rate g/ha</td><td colspan="4"> % Injury (21 DAT)</td>

<td> MARESTAI L (CA) Susceptible</td><td> MARESTAI L (CA) Resistant</td><td> MARESTAI L(KY) Susceptible</td><td> MARESTAI L(KY) Resistant</td>

<td> Roundup WeatherMAX TM</td><td> 840</td><td> 97.2</td><td> 55.0</td><td> 76.7</td><td> 58.3</td>

<td></td><td> 1680</td><td> 100.0</td><td> 64.2</td><td> 97.5</td><td> 79.2</td>

<td></td><td> 2100</td><td> 100.0</td><td> 76.7</td><td> 100.0</td><td> 90.8</td>

<td> Clarity™</td><td> 50</td><td> 68.3</td><td> 61.7</td><td> 78.3</td><td> 78.3</td>

<td></td><td> 140</td><td> 82.5</td><td> 80.8</td><td> 90.0</td><td> 88.3</td>

<td></td><td> 280</td><td> 85.0</td><td> 82.5</td><td> 91.7</td><td> 90.8</td>

Example 7

Development of a method for controlling glyphosate tolerant weeds in a field

Transgenic seeds having dicamba tolerance are planted in a field that has been treated with glyphosate before planting the transgenic seeds. The field is then treated with a herbicidally effective amount of dicamba before or after planting the seeds to control glyphosate resistant weeds. The herbicidally effective amount of dicamba is such that the growth of glyphosate resistant weeds is controlled, but is not injurious to the planted crop as shown in the examples described herein. Thus, transgenic seeds 20 having dicamba tolerance in combination with an effective amount of dicamba are useful for control of glyphosate resistant weeds. The method may be implemented without delaying planting of the dicamba tolerant crop plants, thus providing a significant advance over the prior art, in which dicamba must be applied sufficiently

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517 prior to planting such that the dicamba degrades in the environment sufficiently to avoid injury to crop plants.

Example 8

Combination of dicamba and glyphosate for controlling glyphosate resistant weeds to allow reduced herbicide application rates

As shown in Table 8, dicamba alone was more effective in controlling resistant biotypes at lower application rates than glyphosate. Further, it has unexpectedly been found that dicamba in combination with glyphosate allows control of glyphosate tolerant and susceptible weeds at lower application rates. For example, 10 whereas 200 g/ha of glyphosate was able to control only 6% of marestail (KY resistant biotype) at 18 DAT and 40 g/ha of dicamba was able to control about 52% of the KY biotype at 18 DAT, a 200 g/ha glyphosate and 40 g/ha dicamba mixture was able to control about 79% of the KY biotype at 18 DAT.

In general, any formulation containing dicamba appeared to be more 15 efficacious than glyphosate alone on the resistant biotype. Also, in general, the following trend in effectiveness of glyphosate to dicamba ratio on resistant biotype was found to be true at: 4:1 > 10:1 > 20:1 >40:1 > 80:1. The results show that a glyphosate to dicamba mixture ratio of 4:1 containing 200 g/h glyphosate and 50 g/h dicamba provided superior control than either glyphosate or dicamba alone.

Table 8. Effect of dicamba and glyphosate for controlling glyphosate resistant weeds.

<td rowspan="2"> CHEMICAL FORMULATION</td><td rowspan="2"> Rate g/ha</td><td rowspan="2"> RATIO</td><td colspan="2"> % Injury (18 DAT)</td><td colspan="2"> % Injury (30 DAT)</td>

<td> Marestail Susceptible (KY)</td><td> Marestail Resistant (KY)</td><td> Marestail Susceptible (KY)</td><td> Marestail Resistant (KY)</td>

<td> Roundup WeatherMAX™</td><td> 200</td><td></td><td> 86.0</td><td> 5.8</td><td> 96.3</td><td> 0.0</td>

<td></td><td> 400</td><td></td><td> 99.7</td><td> 25.0</td><td> 100.0</td><td> 18.3</td>

<td></td><td> 800</td><td></td><td> 100.0</td><td> 46.7</td><td> 100.0</td><td> 44.2</td>

<td></td><td> 1600</td><td></td><td> 100.0</td><td> 59.2</td><td> 100.0</td><td> 62.5</td>

CA 02653739 2008-11-28

WO 2007/143690

PCT/US2007/070517

<td rowspan="2"> CHEMICAL FORMULATION</td><td rowspan="2"> Rate g/ha</td><td rowspan="2"> RATIO</td><td colspan="2"> % Injury (18 DAT)</td><td colspan="2"> % Injury (30 DAT)</td>

<td> Marestail Susceptible (KY)</td><td> Marestail Resistant (KY)</td><td> Marestail Susceptible (KY)</td><td> Marestail Resistant (KY)</td>

<td> Clarity™</td><td> 2.5</td><td></td><td> 6.7</td><td> 10.8</td><td> 15.8</td><td> 7.5</td>

<td></td><td> 5</td><td></td><td> 18.3</td><td> 25.0</td><td> 20.8</td><td> 35.0</td>

<td></td><td> 10</td><td></td><td> 34.2</td><td> 35.8</td><td> 29.2</td><td> 39.2</td>

<td></td><td> 20</td><td></td><td> 40.8</td><td> 45.8</td><td> 40.0</td><td> 45.0</td>

<td></td><td> 40</td><td></td><td> 50.0</td><td> 52.5</td><td> 51.7</td><td> 68.3</td>

<td></td><td> 80</td><td></td><td> 68.3</td><td> 69.2</td><td> 71.7</td><td> 84.3</td>

<td></td><td> 100</td><td></td><td> 83.3</td><td> 75.8</td><td> 86.3</td><td> 87.5</td>

<td></td><td> 200</td><td></td><td> 89.2</td><td> 83.3</td><td> 99.3</td><td> 94.3</td>

<td> Roundup WeatherMAX™</td><td> 200 + 2.5</td><td> 80:1</td><td> 50.8</td><td> 20.8</td><td> 55.8</td><td> 31.7</td>

<td> + Clarity™</td><td> 400 + 5</td><td> 80:1</td><td> 85.8</td><td> 39.2</td><td> 97.7</td><td> 40.8</td>

<td></td><td> 800 + 10</td><td> 80:1</td><td> 99.7</td><td> 47.5</td><td> 100.0</td><td> 45.0</td>

<td></td><td> 1600 + 20</td><td> 80:1</td><td> 100.0</td><td> 50.8</td><td> 100.0</td><td> 63.3</td>

<td> Roundup WeatherMAX™</td><td> 200 + 5</td><td> 40:1</td><td> 56.7</td><td> 28.3</td><td> 64.2</td><td> 35.0</td>

<td> + Clarity™</td><td> 400 + 10</td><td> 40:1</td><td> 82.5</td><td> 40.0</td><td> 94.2</td><td> 43.3</td>

<td></td><td> 800 + 20</td><td> 40:1</td><td> 99.3</td><td> 53.3</td><td> 100.0</td><td> 60.8</td>

<td></td><td> 1600 + 40</td><td> 40:1</td><td> 100.0</td><td> 70.8</td><td> 100.0</td><td> 80.8</td>

<td> Roundup WeatherMAX™</td><td> 200 + 10</td><td> 20:1</td><td> 58.3</td><td> 38.3</td><td> 66.7</td><td> 40.0</td>

<td> + Clarity™</td><td> 400 + 20</td><td> 20:1</td><td> 81.7</td><td> 56.7</td><td> 93.3</td><td> 50.0</td>

<td></td><td> 800 +40</td><td> 20:1</td><td> 99.0</td><td> 62.5</td><td> 100.0</td><td> 73.3</td>

<td></td><td> 1600 + 80</td><td> 20:1</td><td> 99.7</td><td> 77.5</td><td> 100.0</td><td> 88.3</td>

<td> Roundup WeatherMAX™</td><td> 200 + 20</td><td> 10:1</td><td> 56.7</td><td> 52.5</td><td> 70.8</td><td> 60.0</td>

CA 02653739 2013-05-10

<td rowspan="2"> CHEMICAL FORMULATION</td><td rowspan="2"> Rate g/ha</td><td rowspan="2"> RATIO</td><td colspan="2"> % Injury (18 DAT)</td><td colspan="2"> % Injury (30 DAT)</td>

<td> Marestail Susceptible (KY)</td><td> Marestail Resistant (KY)</td><td> Marestail Susceptible (KY)</td><td> Marestail Resistant (KY)</td>

<td> + Clarity™</td><td> 400 + 40</td><td> 10:1</td><td> 84.2</td><td> 79.2</td><td> 93.3</td><td> 86.3</td>

<td></td><td> 800 + 80</td><td> 10:1</td><td> 98.7</td><td> 83.3</td><td> 100.0</td><td> 96.8</td>

<td></td><td> 1600 + 160</td><td> 10:1</td><td> 99.7</td><td> 89.2</td><td> 100.0</td><td> 99.3</td>

<td> Roundup WeatherMAX™</td><td> 200 + 50</td><td> 4:1</td><td> 61.7</td><td> 79.2</td><td> 83.5</td><td> 87.2</td>

<td> + Clarity™</td><td> 400 + 100</td><td> 4:1</td><td> 89.2</td><td> 88.3</td><td> 99.7</td><td> 98.7</td>

<td></td><td> 800 + 200</td><td> 4:1</td><td> 99.7</td><td> 88.3</td><td> 100.0</td><td> 99.3</td>

<td></td><td> 1600 + 400</td><td> 4:1</td><td> 100.0</td><td> 89.7</td><td> 100.0</td><td> 100.0</td>

Example 9

Production of transgenic seeds having dicamba and glyphosate tolerance

Methods for producing transgenic seeds having glyphosate tolerance are known in the art and such seeds can be produced by persons of skill in the art by using a polynucleotide encoding glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) as described in U.S. Patent 5,627,061, U.S. Patent 5,633,435, U.S. Patent 6,040,497 and in U.S. Patent 5,094,945, WO04074443 and W004009761.

Soybean breeding lines containing the

Roundup Ready® trait event 40-3-2 (Padgette et al., 1995) have been produced. Seeds from soybean plant designated as MON 19788 have been deposited under ATCC Accession No. PTA-6708.

Glyphosate tolerant plants can also be produced by incorporating polynucleotides encoding glyphosate degrading enzymes such as glyphosate oxidoreductase (GOX, U.S. Patent 5,463,175), a glyphosate-N-actetyl transferase (GAT, U.S. Patent publication 20030083480),

CA 02653739 2013-05-10 and a glyphosate decarboxylase (W005003362; U.S. Patent Application 20040177399).

Dicamba tolerant plants are disclosed herein. A suitable line from each is crossed and progeny seeds screened with herbicide applications of glyphosate and 5 dicamba to obtain progeny expressing both genes and exhibiting tolerance to both dicamba and glyphosate. Alternatively, coding sequences conferring tolerance to one or both of the herbicides are directly introduced into a given Hne. Seeds from these plants are used for developing a method for controlling weed resistance development in a field as described below.

Transgenic seeds having dicamba and glyphosate tolerances were tested for their tolerance to dicamba. glyphosate, or both herbicides. Table 9 shows tolerance of transgenic soybeans carrying glyphosate and dicamba tolerance transgenes to glyphosate, dicamba, and glyphosate and dicamba at various stages of plant growth. Injury was not seen on plants when either or both herbicides were applied at pre15 emergence stage. Post-emergence treatments of either or both herbicides at V3, Rl, and R3-4 showed only little injury.

Table 9. Tolerance of transgenic soybeans carrying glyphosate and dicamba tolerance transgenes to glyphosate, dicamba, and glyphosate and dicamba.

<td> Plant Line</td><td> Herbicide Applied</td><td> ' Rate 1</td><td> Preemergence treatment</td><td colspan="3"> Post-emergence treatment</td>

<td></td><td colspan="2"> ί</td><td></td><td> V3</td><td> Rl</td><td> R3-4</td>

<td></td><td colspan="2"> ; gm ' ae/ha</td><td> 20 DAT</td><td> 8 DAT</td><td> 7 DAT</td><td> 18 DAT</td>

<td></td><td colspan="2"></td><td colspan="4"> % injury (Average of 4 replications)</td>

<td> Non-transgenic Control</td><td> CLARITY</td><td> 561</td><td> 99.0</td><td> 83.8</td><td> 71.3</td><td> 85.0</td>

<td></td><td> RWMax</td><td> 841</td><td> 0.0</td><td> 81.3</td><td> 66.3</td><td> 67.5</td>

<td></td><td> CLARITY+RWMax</td><td> 561+841</td><td> 99.5</td><td> 93.8</td><td> 81.3</td><td> 99.0</td>

<td> RR1+DM0 Line!</td><td> CLARITY</td><td> 561</td><td> 0.0</td><td> 7.0</td><td> 6.3</td><td> 4.5</td>

<td></td><td> RWMax</td><td> 841</td><td> 0.0</td><td> 3.5</td><td> 3.5</td><td> 11.3</td>

CA 02653739 2008-11-28

WO 2007/143690

PCT/US2007/070517

<td></td><td> CLARITY+RWMax</td><td> 561+841</td><td> 0.0</td><td> 3.0</td><td> 4.0</td><td> 10.0</td>

<td> RR1+ DM0 Line 2</td><td> CLARITY</td><td> 561</td><td> 0.0</td><td> 5.3</td><td> 6.3</td><td> 5.3</td>

<td></td><td> RWMax</td><td> 841</td><td> 0.0</td><td> 4.5</td><td> 4.5</td><td> 11.7</td>

<td></td><td> CLARITY+RWMax</td><td> 561+841</td><td> 0.0</td><td> 5.0</td><td> 4.0</td><td> 8.8</td>

<td> RR1+ DM0 Line 3</td><td> CLARITY</td><td> 561</td><td> 0.0</td><td> 9.0</td><td> 8.8</td><td> 7.5</td>

<td></td><td> RWMax</td><td> 841</td><td> 0.0</td><td> 3.5</td><td> 4.0</td><td> 11.3</td>

<td></td><td> CLARITY+RWMax</td><td> 561+841</td><td> 0.0</td><td> 4.5</td><td> 3.5</td><td> 10.0</td>

<td> RR1+ DM0 Line 4</td><td> CLARITY</td><td> 561</td><td> 0.0</td><td> 8.5</td><td> 8.8</td><td> 3.5</td>

<td></td><td> RWMax</td><td> 841</td><td> 0.0</td><td> 3.5</td><td> 3.5</td><td> 11.3</td>

<td></td><td> CLARITY+RWMax</td><td> 561+841</td><td> 0.0</td><td> 4.5</td><td> 4.5</td><td> 8.8</td>

<td> RR2+ DM0 Linel</td><td> CLARITY</td><td> 561</td><td> 0.0</td><td> 8.5</td><td> 6.3</td><td> 5.3</td>

<td></td><td> RWMax</td><td> 841</td><td> 0.0</td><td> 3.5</td><td> 3.5</td><td> 3.0</td>

<td></td><td> CLARITY+RWMax</td><td> 561+841</td><td> 0.0</td><td> 5.0</td><td> 4.5</td><td> 5.0</td>

<td> RR2+ DM0 Line 2</td><td> CLARITY</td><td> 561</td><td> 0.0</td><td> 9.0</td><td> 6.3</td><td> 3.0</td>

<td></td><td> RWMax</td><td> 841</td><td> 0.0</td><td> 3.5</td><td> 6.3</td><td> 3.0</td>

<td></td><td> CLARITY+RWMax</td><td> 561+841</td><td> 0.0</td><td> 9.5</td><td> 7.0</td><td> 3.0</td>

<td> RR2+ DM0 Line 3</td><td> CLARITY</td><td> 561</td><td> 0.0</td><td> 9.5</td><td> 7.5</td><td> 3.5</td>

<td></td><td> RWMax</td><td> 841</td><td> 0.0</td><td> 3.5</td><td> 6.3</td><td> 4.5</td>

<td></td><td> CLARITY+RWMax</td><td> 561+841</td><td> 0.0</td><td> 8.5</td><td> 3.5</td><td> 3.3</td>

<td> RR2+ DM0 Line 4</td><td> CLARITY</td><td> 561</td><td> 0.0</td><td> 5.3</td><td> 5.8</td><td> 3.0</td>

<td></td><td> RWMax</td><td> 841</td><td> 0.0</td><td> 16.5</td><td> 17.0</td><td> 4.0</td>

<td></td><td> CLARITY+RWMax</td><td> 561+841</td><td> 0.0</td><td> 11.0</td><td> 3.5</td><td> 5.3</td>

Example 10

Development of a method for controlling weed resistance development in a field

Transgenic seeds having dicamba and glyphosate tolerance prepared as described above are planted in a field. The field is treated with dicamba and glyphosate before or after planting the seeds using a mixture of dicamba and glyphosate in an effective amount to control weed growth. Typically about a lx

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517 application rate of either herbicide will be effective in controlling weed growth, but the rate may be varied depending upon environmental conditions and the type of weeds being controlled, as is known in the art. The rate of application may also be increased or decreased depending upon the rate of control desired. Generally 5 speaking, increasing the rate of one herbicide will allow a decrease in the rate of the second herbicide in order to obtain the same level of seed control. In specific embodiments, an application of from about 200 to about 1600 g/ha of glyphosate is combined with from about 20 to about 400 g/ha of dicamba.

A desired application rate may be optimized in any particular environment or 10 in the context of a particular weed can be determined using the experimental layout of Example 9 with the different formulation rates described therein. In addition to desired level of weed control, the herbicide level is selected to avoid using more herbicide than is needed on the one hand, and to avoid poor weed control that could lead to herbicide tolerant plants. Over application of herbicides could also damage 15 herbicide tolerant crop. As shown in Example 9 above however, combining optimized applications of these herbicides provides significant levels of control of even herbicide tolerant weeds, and thus represents a major advance in the art.

Example 11

Development of a method for controlling weeds in a single pass in a field

The procedures in Examples 9 and 10 are applied to develop a method for controlling weed growth in a crop-growing environment involving planting a transgenic seed in a field containing a weed or a seed thereof and treating the field in a single pass though the field. The treatment comprises a herbicidally effective 25 amount of dicamba, glyphosate, or a mixture thereof, administered contemporaneously with the planting of the seed. The planting, treating, and growing of the transgenic seed are achieved by standard agricultural methods.

Such a method of planting the transgenic seed and treating the transgenic seed in one pass eliminates the need for a farmer to make multiple passes through the field, 30 including once for planting and once for spraying. The technique therefore reduces fuel and wear-tear costs to farmers.

CA 02653739 2013-05-10

Example 12

Tolerance of plants containing DMO-encoding polynucleotide molecule to other auxin-like herbicides

Herbicide drift and contamination of herbicide delivery equipment is a serious concern in agriculture and can injure non-target crops resulting in losses to farmers. However, some level of drift is often inevitable due to changing environmental conditions such as wind and the proximity of growing fields. Further, it is often difficult and expensive to eliminate all residual levels of a herbicide in a tank following herbicide application and residual herbicides often result in inadvertent injury to crops. Often several rinses of herbicide delivery equipment are required before it can be used for another herbicide, which wastes water and cleaning chemicals.

As herbicides such as 2,4-D and MCPA are post-emergent herbicides for some crops, but can cause serious damage to non-target crops, residual contamination with these herbicides is of particular concern. A transgenic crop tolerant to at least low levels of these herbicides would therefore be of significant value in managing injuries due to spray drift and contamination of herbicide equipment. This could also reduce the extent of equipment washing needed for herbicide delivery equipment.

An analysis was therefore earned out to determine whether soybean plants having DMO-encoding polynucleotide could deactivate other auxin-like herbicides in addition to dicamba, including 2,4-D and MCPA. This was carried out by applying various concentrations of commercially available formulations of other auxin-like herbicides such as 2,4-D (Helena, Collierville, TN), MCPA (Agriliance, St. Paul, MN), triclopyr (GARLON™ 3A; Dow Elanco, Indianapolis, IN), clopyralid (STINGER™; Dow Elanco, Indianapolis, IN), picloram (TORDON™ 22K; Dow Elanco, Indianapolis, IN), or Banvel or CLARITY (BASF, Raleigh, NC) to DMO containing plant tissues or plants.

Transgenic soybean plants were obtained by Agrobacterium-meàiât&amp;à transformation of soybean explants with a DMO-encoding polynucleotide as described above for the events designated Events 1 -4. A non-transgenic line was used as a control. Non-transgenic and transgenic soybean seeds were planted into 3.5-inch square plastic pots containing Redi-earth™ (Scotts-Sierra Horticultural Products Co.,

CA 02653739 2008-11-28

WO 2007/143690

PCT/US2007/070517

Marysville, Ohio). The pots were placed on capillary matting in 35 meh x 60 meh fiberglass watering trays for overhead and/or sub-irrigation for the duration of the test period so as to maintain optimum soil moisture for plant growth. The pots were fertilized with Osmocote (14-14-14 slow release; Scotts-Sierra Horticultural Products Co., Marysville, Ohio) at the rate of 100 gm/cu.ft. to sustain plant growth for the duration of greenhouse trials, and grown in greenhouses at 27°/21°C day/night temperature, with relative humidity between 25%-75% to simulate warm season growing conditions of late spring. A 14 h minimum photoperiod was provided with supplemental light at about 600 μΕ as needed.

All herbicide applications were made with the track sprayer using a Teejet 9501E flat fan nozzle (Spraying Systems Co, Wheaton, IL) with air pressure set at a minimum of 24 psi (165kpa). The spray nozzle was kept at a height of about 16 inches above the top of plant material for spraying. The spray volume was 10 gallons per acre or 93 liters per hectare. Applications were made when plants had reached V-3 stage. All trials were established in a randomized block design (randomized by rate) with 4 to 6 replications of each treatment depending on plant quality, availability and to account for any environmental variability that may have occurred within the confines of each greenhouse.

All treated plants in greenhouse trials were visually assessed at about 4, 14, 18, and 21 days after treatment (DAT) for injury on a scale of 0 to 100 percent relative to untreated control plants, with zero representing “no” injury and 100% representing “complete” injury or death. Data were collected using a palm top computer and analyzed using standard statistical methods. The results shown in Table 10 clearly indicate tolerance of transgenic soybean to other auxin-like herbicides such as 2,4-D and MCPA relative to the non-transgenic line.

Table 10. Percentage injury relative to un-treated controls at 25 DAT post-V3 applications of different auxin-like herbicides to non-transgenic or transgenic soybean plants.*

<td> Herbicide</td><td> Plant/trial</td><td colspan="3"> % injury at shown rates (g ae/ha**) at 21 DAT</td>

<td></td><td></td><td> 280</td><td> 561</td><td> 1120</td>

<td colspan="5"> Dicamba (Clarity)</td>

CA 02653739 2008-11-28

WO 2007/143690

PCT/US2007/070517

<td> Herbicide</td><td> Plant/trial</td><td colspan="6"> % injury at shown rates (g ae/ha**) at 21 DAT</td>

<td></td><td> Nontransgenic</td><td></td><td></td><td> 100</td><td></td><td> 100</td><td></td>

<td></td><td> Event 1</td><td></td><td></td><td> 0.0</td><td></td><td> 1.2</td><td></td>

<td></td><td> Event 2</td><td></td><td></td><td> 0.0</td><td></td><td> 1.7</td><td></td>

<td></td><td> Event 3</td><td></td><td></td><td> 0.0</td><td></td><td> 0.7</td><td></td>

<td></td><td> Event 4</td><td></td><td></td><td> 0.0</td><td></td><td> 1.5</td><td></td>

<td colspan="8"> Dicamba (Banvel)</td>

<td></td><td> Nontransgenic</td><td></td><td></td><td> 100.0</td><td></td><td> 100.0</td><td></td>

<td></td><td> Event 1</td><td></td><td></td><td> 0.0</td><td></td><td> 1.5</td><td></td>

<td></td><td> Event 2</td><td></td><td></td><td> 0.0</td><td></td><td> 0.7</td><td></td>

<td></td><td> Event 3</td><td></td><td></td><td> 0.0</td><td></td><td> 0.5</td><td></td>

<td></td><td> Event 4</td><td></td><td></td><td> 0.0</td><td></td><td> 1.3</td><td></td>

<td colspan="8"> 2,4-D</td>

<td></td><td> Nontransgenic</td><td> 86.8</td><td></td><td> 100.0</td><td></td><td> 100.0</td><td></td>

<td></td><td> Event 1</td><td> 58.3</td><td></td><td> 75.0</td><td></td><td> 100.0</td><td></td>

<td></td><td> Event 2</td><td> 64.2</td><td></td><td> 94.7</td><td></td><td> 100.0</td><td></td>

<td></td><td> Event 3</td><td> 40.0</td><td></td><td> 85.0</td><td></td><td> 100.0</td><td></td>

<td></td><td> Event 4</td><td> 45.8</td><td></td><td> 84.2</td><td></td><td> 100.0</td><td></td>

<td colspan="8"> MCPA</td>

<td></td><td> Nontransgenic</td><td> 93.0</td><td></td><td> 98.3</td><td></td><td> 100.0</td><td></td>

<td></td><td> Event 1</td><td> 72.5</td><td></td><td> 99.3</td><td></td><td> 100.0</td><td></td>

<td></td><td> Event 2</td><td> 55.0</td><td></td><td> 95.0</td><td></td><td> 99.7</td><td></td>

<td></td><td> Event 3</td><td> 55.0</td><td></td><td> 95.8</td><td></td><td> 100.0</td><td></td>

<td></td><td> Event 4</td><td> 88.3</td><td></td><td> 98.8</td><td></td><td> 100.0</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> LSD</td><td> 16.3</td><td></td><td> 10.6</td><td></td><td> 3.7</td><td></td>

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517

<td> Herbicide</td><td> Plant/trial</td><td colspan="6"> % injury at shown rates (g ae/ha**) at 21 DAT</td>

<td colspan="8"></td>

<td></td><td></td><td colspan="6"> % injury shown rates (g ae/ha**) at 14DAT</td>

<td colspan="8"> Triclopyr</td>

<td></td><td> Nontransgenic</td><td> 86.7</td><td></td><td> 97.3</td><td></td><td> 98.7</td><td></td>

<td></td><td> Event 1</td><td> 88.3</td><td></td><td> 95.7</td><td></td><td> 99.3</td><td></td>

<td></td><td> Event 2</td><td> 86.7</td><td></td><td> 98.7</td><td></td><td> 99.3</td><td></td>

<td></td><td> Event 3</td><td> 86.7</td><td></td><td> 94.0</td><td></td><td> 96.3</td><td></td>

<td></td><td> Event 4</td><td> 90.8</td><td></td><td> 98.0</td><td></td><td> 99.2</td><td></td>

<td colspan="8"> Clopyralid</td>

<td></td><td> Nontransgenic</td><td> 99.3</td><td></td><td> 100.0</td><td></td><td> 100.0</td><td></td>

<td></td><td> Event 1</td><td> 99.2</td><td></td><td> 100.0</td><td></td><td> 100.0</td><td></td>

<td></td><td> Event 2</td><td> 98.2</td><td></td><td> 99.7</td><td></td><td> 100.0</td><td></td>

<td></td><td> Event 3</td><td> 99.3</td><td></td><td> 100.0</td><td></td><td> 100.0</td><td></td>

<td></td><td> Event 4</td><td> 99.7</td><td></td><td> 100.0</td><td></td><td> 100.0</td><td></td>

<td colspan="8"> Picloram</td>

<td></td><td> Nontransgenic</td><td> 99.3</td><td></td><td> 100.0</td><td></td><td> 100.0</td><td></td>

<td></td><td> Event 1</td><td> 99.7</td><td></td><td> 100.0</td><td></td><td> 100.0</td><td></td>

<td></td><td> Event 2</td><td> 99.3</td><td></td><td> 100.0</td><td></td><td> 100.0</td><td></td>

<td></td><td> Event 3</td><td> 99.3</td><td></td><td> 99.7</td><td></td><td> 100.0</td><td></td>

<td></td><td> Event 4</td><td> 99.3</td><td></td><td> 100.0</td><td></td><td> 100.0</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> LSD</td><td> 2.9</td><td></td><td> 1.8</td><td></td><td> 1.4</td><td></td>

* The % injury was represented as ANOVA mean comparisons. **grams of active acid equivalent/hectare

Another auxin-like herbicide Butyrac 200 (2,4-DB; Albaugh) was also tested on transgenic soybean plants carrying a DMO gene for testing the plants tolerance to it. The herbicide was applied as a post-emergence treatment at three application rates

CA 02653739 2008-11-28

WO 2007/143690

PCT/US2007/070517 on two transgenic soybean events and compared with a non-transgemc line for total crop injury across all three application rates: 280 g/ha (0.25 lb/a), 561 g/ha (0.5 lb/a) and 841 g/ha (0.75 lb/a) (see Table 11). Both transgenic soybean lines showed low level of tolerance to 2,4-DB. This example shows that dicamba tolerant soybean is also tolerant to low levels of 2,4-DB and should be useful in managing damage from spray drift from the same or neighboring fields to prevent crop loses, and would exhibit tolerance to residual levels of 2,4-DB following incomplete washing of herbicide delivery equipment.

Table 11. Percentage injury relative to the untreated control at 16 DAT by the application of 2,4 -DB to non-transgenic or transgenic soybean plants.

<td> Herbicide</td><td> Plant</td><td colspan="3"> % injury at shown rates (g ae/ha) at 16 DAT</td>

<td></td><td></td><td> 280</td><td> 561</td><td> 1120</td>

<td colspan="5"> 2, 4-DB (Butyrac 200 )</td>

<td></td><td> Non-transgenic NE3001</td><td> 59.2</td><td> 70.0</td><td> 79.2</td>

<td></td><td> 462-1-21</td><td> 25.0</td><td> 43.3</td><td> 75.8</td>

<td></td><td> 469-13-19</td><td> 18.3</td><td> 37.5</td><td> 70.0</td>

This example shows that transgenic soybean plants exhibit tolerance to other auxin-like herbicides, indicating a likely common deactivation mechanism for dicamba and other auxin-like herbicides such as 2,4-D and MCPA. In case of triclopyr, clopyralid, and picloram, the application rate of 280 g ae/ha appeared too stringent in this study and thus lower concentrations may be desired in most settings to reduce plant damage. Thus, a DM0 polynucleotide containing soybean that is tolerant to dicamba is also tolerant to low levels of 2,4-D and MCPA and should prevent or minimize damage from spray drift from same or neighboring fields to prevent crop loses, and would exhibit tolerance to residual levels of these herbicides following incomplete washing of herbicide delivery equipment. The herbicide delivery equipment could include a tank, container, hose, strainer, boom, sprayer, nozzle, pump, and accessories such as coupling, elbows, shanks, and valves. The delivery équipement is operable manually or mechanically for example on a farm vehicle, airplane, and helicopter, among others.

CA 02653739 2013-05-10

Example 13

Production of dicamba tolerant transgenic corn plants

To test the use of a DMO gene in providing dicamba tolerance to monocots, transgenic com plants were produced that comprise a DMO gene as disclosed above with or without a transit peptide (e.g. TaWaxy, CTP1, CTP2synthetic, CTP4) under the control of plant gene expression elements such as a promoter (e.g. PC1SV, e35S, OsActl, OsTPl, OsActl5), and an intron (e.g. OsActl, OsActl5, OsTPl, ZmHSP70). This expression element contains first intron and flanking UTR exon sequences from the rice actin 1 gene and includes 12 nt of exon 1 at the 5' end and 7 nt of exon 2 at the 3' end), and a 3’UTR (e.g. TaHspl7). Nucleotide sequences/ and or patent references for various expression elements are disclosed in co-pending application U.S. Ser. No. 60/891,675.

Transgenic com plants were produced by the methods known in the art such as WO9506722 and US patent application 20040244075. Transgenic com events having single copy were evaluated for dicamba tolerance at a single location replicated trial. Six events from each of the six constructs were used. The experimental design was as follows: rows/entry: 1; treatment: 0.5 lb/'a of dicamba at V3 stage followed by 1 lb/a of dicamba at V8 stage (Clarity®, BASF, Raleigh, NC); replications: 2; row spacing: 30 inches; plot length: minimum 20 feet; plant density: about 30 plants/17.5 ft.; alleys: 2.5 feet. The entire plot was fertilized uniformly to obtain an agronomically acceptable crop. A soil insecticide such as Force® 3G (Syngenta Crop Protection, Greensboro, NC, USA) at 5 oz. per 1000 ft. of row for control of com rootworm was applied at planting time. If black cutworm infestation was observed, POUNCE® 3.2EC at 4 to 8 oz. per acre rate (FMC Corporation, Philadelphia, PA) was used. In addition, an insecticide spray program was used to control all above ground lepidopteran pests including European com borer, com earworm, and fall armyworm. POUNCE® 3.2EC at 4 to 8 oz. per acre was applied ever}' 3 weeks to control lepidopteran pests; about 4 applications were made. The plot was kept weed free with a pre-emergence application of a herbicide such as Hamess® Xtra 5.6L (Monsanto, St. Louis, MO) and Degree Xtra® (Monsanto, St. Louis, MO). If weed escapes were observed in the untreated check, they were controlled by hand weeding or a postemergence application of PERMIT™ (Monsanto, St. Louis, MO) or BUCTRIL (Bayer, Research Triangle Park. NC) over the entire trial.

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517

Com inbred lines transformed with DNA constructs comprising a DMO transgene were tested for dicamba tolerance by measuring brace root injury when treated with 0.5 lb/a of dicamba at V3 stage followed by 1 lb/a of dicamba at V8 stage. Brace root injury was evaluated visually by counting the number of plants in 5 a row showing an “atypical” morphology of having the brace roots fused as compared to a typical morphology of “finger-like” structure. As shown in Table 12, com plants transformed with DNA constructs coding for a DMO without linking it to a CTP (pMON73699, pMON73704) showed higher level of brace root injury, i.e. lower level of protection upon dicamba treatment. The constructs coding for a 10 DMO linked to a CTP (pMON73716, pMON73700, pMON73715, pMON73703) showed lower level of brace root injury, i.e. higher level of protection upon dicamba treatment.

Table 12. Percentage brace root injury as a measure of dicamba tolerance exhibited by transgenic corn plants transformed with DNA constructs carrying 15 DMO.

<td> Inbreds/Constructs</td><td> Details</td><td> Brace root injury</td>

<td> 01CSI6</td><td> Susceptible inbred to dicamba</td><td> 95.4</td>

<td> LH244</td><td> Resistant inbred to dicamba</td><td> 93.8</td>

<td> pMON73699</td><td> PC 1 SV/I-OsActl/DMO-Wmc/TaHsp 17</td><td> 93.2</td>

<td> pMON73704</td><td> e35S/I-OsActl/DMO-Wmc/TaHsp 17</td><td> 91.3</td>

<td> pMON73716</td><td> PC 1 SV/I-OsActl/TaWaxy/DMO- Wmc/TaHspl7</td><td> 78.8</td>

<td> pMON73700</td><td> PClSV/I-OsActl/CTPl/DMO- Wmc/TaHspl7</td><td> 74.4</td>

<td> pMON73715</td><td> PClSV/I-OsActl/CTP2syn/DMO- Wmc/TaHspl7</td><td> 68.2</td>

<td> pMON73703</td><td> e35S/I-OsActl/CTPl/DMO-Wmc/TaHspl7</td><td> 68.8</td>

Example 14

Production of dicamba tolerant transgenic cotton plants

To test the use of DMO gene in providing dicamba tolerance to cotton, transgenic cotton plants were produced. Several DNA constructs carrying a DMO 20 coding region as disclosed herein with a transit peptide (e.g., PsRbcS CTP, CTP1, CTP2) under the control of plant gene expression elements such as a promoter (e.g. PC1SV, FMV, or e35S), and a 3’UTR (e.g. E6; Accession # U30508) were produced and transformed into cotton (Gossypium hirsutum) as follows. Nucleotide sequences/

CA 02653739 2008-11-28

WO 2007/143690

PCT/US2007/070517 and or patent refemces for various expression elements are disclosed in co-pending application U.S. Ser. No. 60/891,675. Media used are noted in Table 13.

Cotton transformation was performed, for instance as described according to U.S. Patent Application Publication 20040087030, via an embryogénie approach.

Expiants of cotton cv Coker 130 were grown in vitro and with a liquid suspension of Agrobacterium tumefaciens carrying a DNA construct of interest, using selection on kanamycin containing media. Putative transgenic plantlets were then transferred to soil to obtain mature cotton plants. The transgenic nature of transformants was confirmed by DNA testing.

f0 Table 13. Composition of various media used for cotton transformation.

<td> Components</td><td colspan="5"> Amount/L</td>

<td></td><td> Glucose</td><td> Sucrose</td><td> UMO</td><td> TRP+</td><td> SHSU</td>

<td> MS basal salts (Phytotech.)</td><td> 4.33 g</td><td> 4.33 g</td><td> 4.33 g</td><td> 4.33 g</td><td> -</td>

<td> Gamborg’s B5 vitamins (Phytotech) (500X)</td><td> 2 ml</td><td> 2 ml</td><td> 2 ml</td><td> 2 ml</td><td> -</td>

<td> 2, 4-D (1 mg/ml)</td><td> 0.1 ml</td><td> 0.1 ml</td><td> -</td><td></td><td> -</td>

<td> Stewart and Hsu majors (10X)</td><td> -</td><td> -</td><td> -</td><td> -</td><td> 100 ml</td>

<td> Stewart and Hsu minors (100X)</td><td> -</td><td> -</td><td> -</td><td> -</td><td> 10 ml</td>

<td> Steward and Hsu organic (100X)</td><td> -</td><td> -</td><td> -</td><td> -</td><td> 10 ml</td>

<td> Kinetin (0.5 mg/ml)</td><td> 1 ml</td><td> 1 ml</td><td> -</td><td> -</td><td> -</td>

<td> Chelated iron (100X)</td><td> -</td><td> -</td><td></td><td></td><td> 1.5 ml</td>

<td> Glucose</td><td> 30 g</td><td> 30 g</td><td> 30 g</td><td> 30 g</td><td> 5g</td>

<td> Potassium nitrate</td><td> -</td><td> -</td><td> -</td><td> 1.9 g</td><td> -</td>

<td> Casein hydrolysate</td><td> -</td><td> -</td><td> -</td><td> o.l g</td><td> -</td>

<td> pH</td><td> 5.8</td><td> 5.8</td><td> 5.8</td><td> 5.8</td><td> 6.8</td>

<td> Phytagel (Sigma)</td><td> 2.5 g</td><td> 2.5 g</td><td> -</td><td> -</td><td> -</td>

<td> Gelrite (Kelco)</td><td> -</td><td> -</td><td> 3.5 g</td><td> 3.5 g</td><td> 2.2 g</td>

<td> Carbenicillin (250 mg/ml)</td><td> 1.7 ml</td><td> 1.7 ml</td><td> 1.7 ml</td><td> 1.7 ml</td><td></td>

<td> Cefotaxime (100 mg/ml)</td><td> 1 ml</td><td> 1 ml</td><td> 1 ml</td><td> 1 ml</td><td> -</td>

<td> Benlate (50 mg/ml)</td><td> -</td><td> -</td><td> -</td><td> 1 ml</td><td> 1 ml</td>

<td> Kanamycin (50 mg/ml)</td><td> 0.8-1.0 ml</td><td> 0.8-1.0 ml</td><td> 1 ml</td><td> -</td><td> -</td>

CA 02653739 2013-05-10

<td> Sucrose</td><td> -</td><td> 0.1 g</td><td> -</td><td> -</td><td> -</td>

<td> Ascorbic acid</td><td> -</td><td> -</td><td> too mg</td><td></td><td></td>

Transformed cotton plants that comprise a DNA construct, i.e, each comprising a different combination of a DM0 coding region with a transit peptide, a promoter, and a 3’UTR, were treated with dicamba (Clarity®, BASF, Raleigh, NC) as a post-emergent treatment at V4-5 growth stage at the rate of 561 g ae/ha (0.5 Ib/a) and found to be tolerant whereas untransformed cotton plants showed an injury rate of 79% to 86%. Transgenic plants showing more than 95% tolerance (equal to less than 5% injury) were selected for further studies. Transgenic plants were also tolerant to a subsequent post-emergent treatment of dicamba. For example, the plants that were treated with 0.5 lb/acre of dicamba at V3-4 stage followed by either 1 or 2 lb/acre of dicamba at V5 or later stages were still tolerant to dicamba. R1 transgenic seeds and plants were also subjected to pre-emergence or pre-emergence and post-emergence dicamba treatment and found to be tolerant. This example shows that a DM0 gene can provide dicamba tolerance to cotton at various stages of growth thus enabling application of dicamba at various stages to obtain effective weed control.

* * * * * *

AU of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. The scope of the claims should not be limited by the preferred embodiments set forth herein, but should be given the broadest interpretation consistent with the description as a whole.

CA 02653739 2013-05-10

REFERENCES

The references listed below are referred to herein to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and/or compositions employed herein.

U.S. Patent 4,554,101; U.S. Patent 5,004,863; U.S. Patent 5,015,580; U.S. Patent 5,017,692; U.S. Patent 5,094,945; U.S. Patent 5,159,135; U.S. Patent 5,322,938; U.S.

Patent 5,352,605; U.S. Patent 5,359,142; U.S. Patent 5,384,253; U.S. Patent 5,424,412; U.S. Patent 5,445,962; U.S. Patent 5,463,175; U.S. Patent

5,508,184; U.S. Patent 5,530,196; U.S. Patent 5,538,880; U.S. Patent

5,550,318; U.S. Patent 5,552,299; U.S. Patent 5,567,600; U.S. Patent

5,567,862; U.S. Patent 5,591,616; U.S. Patent 5,627,061; U.S. Patent

5,633,435; U.S. Patent 5,633,437; U.S. Patent 5,635,055; U.S. Patent

5,641,876; U.S. Patent 5,689,052; U.S. Patent 5,750,871; U.S. Patent

5,824,877; U.S. Patent 5,837,848; U.S. Patent 5,846.797; U.S. Patent

5,859,347; U.S. Patent 5,939,602; U.S. Patent 5,981,840; U.S. Patent

6,040,497; U.S. Patent 6,140,078; U.S. Patent 6,160,208; U.S. Patent

6,175,060; U.S. Patent 6,177,611; U.S. Patent 6,232,526; U.S. Patent

6,252,138; U.S. Patent 6,294,714; U.S. Patent 6,384,301; U.S. Patent

6,388,170; U.S. Patent 6,399,861; U.S. Patent 6,403,865; U.S. Patent

6,414,222; U.S. Patent 6,426,446; U.S. Patent 6,429,357; U.S. Patent

6,429,362; U.S. Patent 6,433,252; U.S. Patent 6,437,217; U.S. Patent

6,613,963; U.S. Patent 6,635,806; U.S. Pat. No. 6,677,503; U.S. Patent 7,132,528

U.S. Appln. Serial 09/757,089

U.S. Patent Appln. Publication 20030083480

U.S. Patent Appln. Publication 20030135879

U.S. Patent Appln. Publication 2004087030

U.S. Patent Appln. Publication 20070079393

U.S. Provisional Patent Appl. Ser. No. 60/891,675

Anonymous, Greenbook Crop Protection Reference, 23' edition, Greenbook Products. Lenexa, KS, 2007.

Becker et al., Plant Mol. Biol., 20(1):49-60, 1992.

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517

Behrens et al., Science 316:1185-1188, 2007.

Buchanan-Wollaston et al., J. Cell. Biochem., Supp. 13D, 330, 1989.

Chandler et al., Plant Cell, 1:1175-1183, 1989.

Chandler, In: CRC Handbook of Pest Management in Agriculture, Pimentel (Ed.), 1:95-109, 1981.

Cornai et al., Nature, 317:741, 1985.

Cork and Khalil, Adv. Appl. Microbiol., 40:289-321, 1995.

Cork and Krueger, Adv. Appl. Microbiol., 36:1-66, 1991.

Coruzzi et al., EMBOJ., 3: 1671, 1984.

Creissen et al., Plant J., 8(2):167-175, 1995.

Crop Protection Chemicals Reference, Chemical &amp; Pharmaceutical Press, Inc., NY, 11^th Ed., 1803-1821, 1995

De Block et al., EMBOJ., 6(9):2513-2518, 1987.

della-Cioppa et al., Proc. Natl. Acad. Sci. USA, 83:6873-6877, 1986.

Ebert et al., Proc. Natl. Acad. Sci. USA, 84:5745-5749, 1987.

European Appln. 553494

European Appln. 646643

Klee et al., Mol. Gen. Genet., 210:437-442, 1987.

Kyte and Doolittle, J. Mol. Biol., 157(1):105-132, 1982.

Lawton et al., Plant Mol. Biol. 9:315-324, 1987.

Mazur, et al., Nucleic Acids Res., 13(7):2373-2386, 1985.

Miki et al., In: Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson (Eds.), CRC Press, 67-88, 1993.

Misawa et al, Plant J., 4:833-840, 1993.

Misawa et al, Plant J., 6:481-489, 1994.

Odell et al., Nature, 313:810-812, 1985.

Padgette et al., Crop Sci., 35:1451-1461, 1995.

PCT Appln. WO 95/06722

PCT Appln. WO 97/41228

PCT Appln. WO 96/38567

PCT Appln. WO 97/31115

PCT Appln. WO 97/11086

PCT Appln. WO 04009761

PCT Appln. WO 04074443

CA 02653739 2008-11-28

WO 2007/143690 PCT/US2007/070517

Sathasiivan et al., Nucl. Acids Res., 18:2188-2193, 1990.

Stalker et al., Science, 242:419, 1988.

Stalker et al., Science, 242:419-422, 1988.

Streber and Willmitzer, Bio/Technology, 7:811, 1989.

VanGessel and Majek, 2005 Soybean Weed Management Guide: for Delaware and New

Jersey, University of Delaware and Rutgers University, 2005.

Walker et al., Proc. Natl. Acad. Sci. USA, 84:6624, 1987.

Yang and Russell, Proc. Natl. Acad. Sci. USA, 87:4144-4148, 1990.

Zhang et al. Plant Cell, Tissue and Organ Culture 56: 37-46, 1999.