WO2013016210A1 - Methods and compositions to produce rice resistant to accase inhibitors - Google Patents

Abstract

Mutant rice resistant/tolerant to ACCase inhibiting herbicides, in particular FOP herbicides, are listed in Table 1. The ACCase inhibitng herbicides used for selection include quizalofop. An exemplary mutant rice tolerant to an ACCase herbicide is disclosed, with a rice genome having G2096S or the equivalent, in the carboxyl transferase domain of the ACCase coding gene, using the Black-Grass numbering system. This mutation shows differential response to FOPs vs. DIMs herbicides, and a greater differential with comparable non-resistant rice lines. Methods to control weeds and methods to produce herbicide resistant rice including transgenic rice, are disclosed.

Description

METHODS AND COMPOSITIONS TO PRODUCE

RICE RESISTANT TO ACCASE INHIBITORS

Inventors: Melissa Hinga, Steven Griffin, Shannon Moon, Russell D. Rasmussen, and

Federico Cuevas

BACKGROUND

[0001] This application claims priority from U.S. Provisional Application No.

61/510,585 filed July 22, 201 1 and U.S. Provisional Application No. 61/541 ,832 filed September 30, 2011, both incorporated by reference. Novel rice plants are described and disclosed that are characterized by tolerance/resistance to herbicides that are ACCase inhibitors and exhibit other characteristics beneficial to rice crops. Methods to control weeds by use of herbicide resistant rice in fields, and methods to produce herbicide resistant rice using e.g. transgenes encoding for a mutant ACCase enzyme, are also disclosed.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been

submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on July 20, 2012, is named

115760_SEQ_ST25.txt and is 32,768 bytes in size.

Value of Rice Crops

[0003] Rice is an ancient agricultural crop and is today one of the principal food crops of the world. There are two cultivated species of rice: Oryza sativa L, the Asian rice, and Oryza glaberrima Steud. , the African rice. The Asian species constitutes virtually all of the world's cultivated rice and is the species grown in the United States. Three major rice producing regions exist in the United States: the Mississippi Delta (Arkansas, Mississippi, northeast Louisiana, southeast Missouri), the Gulf Coast (southwest Louisiana, southeast Texas), and the Central Valleys of California.

[0004] Rice is one of the few crops that can be grown in a shallow flood as it has a unique structure allowing gas exchange through the stems between the roots and the atmosphere. Growth in a shallow flood results in the best yields and is the reason that rice is usually gown in heavy clay soils or soils with an impermeable hard pan layer just below the soil surface. These soil types are usually either not suitable for other crops or at the best the crops yield poorly. [0005] The constant improvement of rice is imperative to provide necessary nutrition for a growing world population. A large portion of the world population consumes rice as their primary source of nutrition. Rice improvement is carried out through conventional breeding practices and by recombinant genetic techniques. Though appearing straight forward to those outside this discipline, crop improvement requires keen scientific and artistic skill.

[0006] Although specific breeding objectives vary somewhat in the different rice producing regions, increasing yield is a primary objective in all programs.

[0007] Plant breeding begins with the analysis and definition of strengths and weaknesses of the current cultivars, followed by the establishment of program goals, to address the latter including the definition of specific breeding objectives. The goal is to combine in a single cultivar an improved combination of desirable traits from the parental sources. These important traits may include higher yield, resistance to environmental stress, diseases and insects, better stems and roots, tolerance to low temperatures, better agronomic characteristics, and grain quality.

[0008] The breeder initially selects and crosses two or more parental lines,

followed by selection among the many new genetic combinations. The breeder can theoretically generate billions of new and different genetic combinations via crossing. The breeder has no direct control at the cellular level; therefore, two breeders will never develop the same line, or even very similar lines, having the same rice traits.

[0009] Pedigree breeding is used commonly for the improvement of self- pollinating crops such as rice. Two parents which possess favorable,

complementary traits are crossed to produce an Fi generation. One or both parents may themselves represent an Fi from a previous cross. Subsequently a segregating population is produced, growing the seeds resulting from selfing one or several F] S if the two parents are pure lines, or by directly growing the seed resulting from the initial cross if at least one of the parents is an Fj . Selection of the best individuals may begin in the first segregating population or F₂; then, beginning in the F3, the best individuals in the best families are selected. "Best" is defined according to the goals of a particular breeding program e.g., to increase yield, resist diseases.

Overall a multifactorial approach is used to define "best" because of genetic interactions. A desirable gene in one genetic background may differ in a different background. In addition, introduction of the gene may disrupt other favorable genetic characteristics. Replicated testing of families can begin in the F4 generation to improve the effectiveness of selection for traits with low heritability. At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines or mixtures of phenotypically similar lines are tested for potential release as new parental lines.

Backcross breeding has been used to transfer genes for a highly heritable trait into a desirable homozygous cultivar or inbred line which is the recurrent parent. The source of the trait to be transferred is called the donor parent. The resulting plant is expected to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait transferred from the donor parent. After the initial cross, individuals possessing the phenotype of the donor parent are selected and repeatedly crossed (backcrossed) to the recurrent parent. The process is used to recover all of the beneficial characteristics of the recurrent parent with the addition of the new trait provided by the donor parent.

Promising advanced breeding lines are thoroughly tested and compared to appropriate standards in environments representative of the commercial target area(s) for at least three or more years. The best lines are candidates for new commercial varieties or parents of hybrids; those still deficient in a few traits may be used as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing and distribution, usually take from 8 to 12 years from the time the first cross is made and may rely on the development of improved breeding lines as precursors. Therefore, development of new cultivars is a time-consuming process that requires precise forward planning, efficient use of resources, and a minimum of changes in direction.

The improvement of rice through breeding is restricted to the natural genetic variation in rice and hybridizing species, such as wild rice. The introduction of new variation in a breeding program is usually through the crossing program as described, such as pedigree or backcross breeding. However, occasionally natural mutations are found that result in the introduction of new traits such as disease resistance or height changes. Breeders have also developed new traits by inducing mutations (small changes in the DNA sequence) into a rice genome. Commonly, EMS or sodium azide plus MNU are used as mutagenic agents. These chemicals randomly induce single base changes in DNA, usually of G and C changed to A and T. Most of these changes have no effect on the crop as they fall either outside the gene coding regions or don't change the amino acid sequence of the gene product.

[00014] The breeder has no direct control of mutation sites in the DNA sequence.

The identification of useful changes is due to the random possibility that an effective mutation will be induced and that the breeder will be able to select that mutation. Seeds are treated with the mutagenic chemical and immediately planted to grow and produce M2 seed. The M2 seed will carry numerous new variations; therefore, no two experiments will produce the same combinations. Among these variations new traits previously not existing in rice and unavailable for selection by a plant breeder may be found and used for rice improvement.

[00015] To find new traits the breeder must use efficient and strategic selection strategies as the process is completely random and has an extremely low frequency of useful new combinations. Among thousands of induced new genetic variants there may be only one with a desirable new trait. An optimal selection system will screen through thousands of new variants and allow detection of a few or even a single plant that might carry a new trait. After identifying or finding a possible new trait the breeder must develop a new cultivar by pedigree or backcross breeding and extensive testing to verify the new trait and cultivar exhibits stable and heritable value to rice producers.

[00016] Using recombinant genetic techniques, nucleic acid molecules with

mutations, that encode improved characteristics in rice, may be introduced into rice with commercially suitable genomes. After a mutation is identified, it may be transferred into rice by recombinant techniques.

Applications of Herbicide Resistance Patents in Rice

[00017] Weeds in crops compete for resources and greatly reduce the yield and quality of the crop. Weeds have been controlled in crops through the application of selective herbicides that kill the weeds but do not harm the crop. Usually selectivity of the herbicides is based on biochemical variations or differences between the crop and the weeds. Some herbicides are non-selective, meaning they kill all or almost all plants. Non-selective or broad spectrum herbicides can be used in crops if new genes are inserted that express specific proteins that convey tolerance or resistance to the herbicide. Resistance to herbicides has also been achieved in crops through genetic mutations that alter proteins and biochemical processes. These mutations may arise in nature, but mostly they have been induced in crops or in vitro in tissue cultures. Unfortunately in some instances, especially with repeated use of a particular herbicide, weeds have developed resistance through the unintended selection of natural mutations that provide resistance. When weeds become resistant to a particular herbicide, that herbicide is no longer useful for weed control. The development of resistance in weeds is best delayed through alternating the use of different modes of action to control weeds, interrupting development of resistant weeds.

[00018] Rice production is plagued by a particularly hard to control weed called red rice. The difficulty arises because red rice is so genetically similar to cultivated rice (they occasionally cross pollinate) that there are no selective herbicides available that target red rice, yet do not harm the cultivated rice. Control is currently provided in commercial rice production through the development of mutations found in rice that render rice resistant to broad spectrum herbicides e.g. imidazolinone and sulfonylurea herbicides.

[00019] Finding new mutations in rice that makes it resistant to herbicides, and to combinations of herbicides with alternative modes of action would greatly benefit rice production. Obtaining and incorporating genes for herbicide resistance into rice genomes with additional favorable characteristics and alternative resistances is challenging, unpredictable, time consuming and expensive, but necessary to meet the world's increasing food needs.

SUMMARY

[00020] Described herein are distinctive rice lines with unique resistances to

herbicides with alternative modes of action. These rice lines should extend the useful life of several herbicides due to being able to rotate the kinds of herbicides applied in grower's fields thus slowing the development of weed resistance.

Several methods are possible to deploy these resistances into hybrids or varieties for weed control, as well as options for hybrid seed production. The rice lines described herein represent new methods for weed control in rice and can be deployed in any of many possible strategies to control weeds and provide for long- term use of this and other weed control methods. In particular, mutant rice tolerant to ACCase inhibiting herbicides is disclosed. These are plants with defined amino acid sequences. [00021] For example, rice with the ACCase mutant G2096S is already

agronomically adapted and through breeding or backcrossing as described herein, will provide herbicide resistance in commercially suitable biological material.

[00022] A mutant rice tolerant to an ACCase inhibitor herbicide is disclosed that has a mutation G2096S in the carboxyl transferase coding region of the ACCase gene, using the Black Grass {Alopecurus myosuroides) numbering system. The mutation makes the acetyl-coenzyme A carboxylase enzyme tolerant/resistant to ACCase inhibitors used as herbicides.

[00023] Cells derived from herbicide resistant seeds, plants grown from such seeds and cells derived from such plants, progeny of plants grown from such seed and cells derived from such progeny are within the scope of this disclosure. The growth of plants produced from deposited seed, and progeny of such plants will typically be resistant/tolerant to acetyl-Coenzyme A carboxylase-inhibiting herbicides at levels of herbicide that would normally inhibit the growth of a corresponding wild-type plant.

[00024] A method for controlling growth of weeds in vicinity to rice plants is also within the scope of the disclosure. One example of such methods is applying one or more herbicides to the weeds and to the rice plants at levels of herbicide that would normally inhibit the growth of a rice plant. For example, at least one herbicide inhibits acetyl-Coenzyme A carboxylase activity. Such methods may be practiced with any herbicide that inhibits acetyl-Coenzyme A carboxylase activity and any resistant rice mutation, e.g., the three embodiments disclosed herein.

[00025] A method for growing herbicide-tolerant rice plants include (a) planting resistant rice seeds; (b) allowing the rice seeds to sprout; (c) applying one or more herbicides to the rice sprouts at levels of herbicide that would normally inhibit the growth of a rice plant. For example, at least one of the herbicides inhibits acetyl- Coenzyme A carboxylase. Such methods may be practiced with any herbicide that inhibits acetyl-Coenzyme A carboxylase activity.

[00026] Methods of producing herbicide-tolerant rice plants that may also use a transgene. One example of such a method is transforming a cell of a rice plant with a transgene, wherein the transgene encodes an acetyl-Coenzyme A carboxylase enzyme that confers tolerance in resulting rice plant to at least one herbicide selected from the group consisting of aryloxyphenoxypropionate herbicides, cyclohexanedione herbicides, phenypyrazoline herbicides or combinations thereof. Any suitable cell may be used in the practice of these methods, for example, the cell may be in the form of a callus. An embodiment of a transgenic is one comprising a mutation in a nucleic acid encoding ACCase, from G to S in position 2096 (Black Grass numbering system).

[00027] A recombinant, mutagenized, synthetic, and/or isolated nucleic acid

molecule including a nucleotide sequence encoding a mutagenized acetyl- Coenzyme A carboxylase of a plant rice, in which the amino acid sequence of the mutagenized acetyl-Coenzyme A carboxylase differs from an amino acid sequence of an acetyl-Coenzyme A carboxylase of the corresponding wild-type plant, are within the scope of the disclosure.

[00028] Different mutations in the ACCase encoding gene are often associated with resistance to specific types of ACCase inhibiting herbicides (FOPS), (DIMS). The specificity of different mutations thus offers the possibility of developing multiple modes of action for weed control in rice.

BRIEF DESCRIPTION OF DRAWINGS

[00029] FIG. 1 shows surviving plants in quizalofop sprayed row 1 1 USAG51477 marked with a flag.

[00030] FIG. 2 shows quizalofop survivors following transplanting.

[00031] FIG. 3 shows a 1 1USAG52084-2 rice plant with seed set.

[00032] FIG. 4 shows DNA sequence for the carboxyl transferease coding region in the ACCase coding gene; a single nucleotide change from G2096S is identified in the mutant line ML0831265-0149 which is identified as 09PM72399. FIG. 4 discloses SEQ ID NOS 2-4, respectively, in order of appearance.

[00033] FIG. 5 shows comparison of protein sequences for the carboxyl transferase region of the ACCase gene; line with code 09PM72399 is the line ML0831265- 0149; this line shows a change of a single amino acid at position 2096, relative to Black-Grass; R0146 is the original line treated with a mutagen to produce the mutation population. FIG. 5 discloses SEQ ID NOS 5-7, respectively, in order of appearance.

[00034] FIG. 6 shows results of plants from ML0831265-0149 at different

application rates of quizalofop herbicide.

[00035] FIG. 7 shows results of plants from ML0831265-0149 with application of different ACCase type herbicides. [00036] FIG. 8 is the mutant nucleotide sequence (SEQ ID NO: 1) that encodes an

ACCase enzyme with S instead of G at position 2096 (Black Grass number system).

[00037] FIG. 9 shows a comparison of % injury after contact with FOP's ACCase inhibiting herbicides in (a) rice plants with a G2096S mutation; and (b) rice plants without the mutation, with a published comparison for a different mutation.

DETAILED DESCRIPTION

[00038] Rice, Oryza sativa L., is an important and valuable field crop. Thus, a continuing goal of rice breeders is to develop stable and high yielding rice cultivars that are agronomically sound. Growers are constantly expecting increasing yields from new varieties and hybrids as a way to increase their economic condition. In addition on a population level increasing yields is necessary due to expanding nutritional needs but limited production resources. To accomplish this goal, the rice breeder must select and develop rice plants possessing required traits and superior yields.

[00039] Acetyl-Coenzyme A carboxylase (ACCase; EC 6.4.1.2) enzymes

synthesize malonyl-CoA as the start of the de novo fatty acid synthesis pathway in plant chloroplasts. ACCase in grass chloroplasts is a multifunctional, nuclear- genome-encoded, very large, single polypeptide, transported into the plastid via an N-terminal transit peptide. The active form in grass chloroplasts is a homodimeric protein.

[00040] ACCase enzymes in grasses are inhibited by three classes of herbicidal active ingredients. The two most prevalent classes are aryloxyphenoxypropanoates ("FOPs") and cyclohexanediones ("DIMs"). In addition to these two classes, a third class phenylpyrazolines ("DENs") has been described.

[00041] Certain mutations in the carboxyl transferase region of the ACCase enzyme results in grasses becoming resistant to ACCase herbicides. In the weed Black- Grass at least five mutations have been described which provide resistance to FOP or DIM class of ACCase herbicides. Some mutations rendering ACCase enzymes resistant to these herbicides may be associated with decreased fitness.

Mutation Population and Establishment

[00042] A mutation breeding program was initiated to develop proprietary herbicide tolerant lines. A permanent mutant population was created by exposing approximately 10,000 seeds (estimated by the average weight of a kernel) of three lines including P1003, R0146, and P1062 to mutagens sodium azide (AZ) and methyl-nitrosourea (MNU). The treated seeds were space planted. Individual plants were harvested creating 8,281 mutation lines. The mutation lines have been maintained as a permanent mutant population for trait screening.

Herbicide Screening

[00043] The permanent mutant population was screened with quizalofop herbicide.

Applicants planted 2,735 M2 progeny rows from R0146, 3,774 M2 progeny rows from P1003 and 655 M2 progeny rows from P1062 in two replications with an estimated 250,000 plants total in each replication. Quizalofop was applied with a rate of 15 oz/acre (1 15.59 gmai/ha) to the first replication 27 days after planting. Plants were at the 3-4 leaf stage and were actively growing when herbicide was applied. The field was flushed the day after application. After about 9 days surviving plants were found in four different progeny rows showing an estimated mutation rate of 0.006% (FIGS. 1-3).

Protein Sequence Comparison on Lines Showing Resistance to ACCase Herbicides

[00044] A portion of the gene that codes for the plastidic ACCase protein was sequenced from all the plants that survived application of quizalofop. Only the carboxyl transferase coding region of the gene was sequenced (FIG. 4). All of the plants deriving from line ML0831265-01493 had a mutation in the DNA sequence that caused an amino acid change in the ACCase protein. One DNA base was changed in the codon for amino acid 2096 relative to numbering in Black-Grass. (Gen Bank CAC84161.1 , denoted as "Am") (FIG. 5). In both rice and Black-Grass the amino acid at position 2096 is glycine. A resistant mutation in Black-Grass changes the amino acid at this position to alanine. The mutation found in rice surviving quizalofop application with designation ML0831265-01493 changes the DNA codon for amino acid 2096 from GGC to AGC causing a serine to be inserted in position 2096 instead of glycine. The mutant line showed resistance to quizalofop in the first screening of the mutant population. Later screening with quizalofop confirmed the resistance to FOPs. The surviving mutant lines were susceptible to DIM type ACCase inhibiting herbicides.

[00045] None of the other lines from the mutant population that survived screening with quizalofop carried a mutation in the carboxyl transferase region of the ACCase coding gene however they have been confirmed to carry resistance to quizalofop herbicide. The resistance in these lines is likely derived from changes outside the carboxyl transferase region of ACCase or could be derived from a different type of resistant mechanism

Resistant Plants

[00046] After herbicide treatment, the surviving plants before transplanting were green and healthy looking whereas all surrounding plants within the row and in adjacent rows were dead. The plants after transplanting were maintained and harvested. The progeny were maintained, tested, and developed as a source of herbicide resistance in production rice (Table 1). This trait is backcrossed or bred into proprietary rice lines and used to develop new varieties or hybrids that will provide producers with an alternative mode of action to control weeds in rice. Affording this opportunity to growers is of great value both in providing high yields and in extending the useful life of currently used weed control technologies. These herbicide resistant lines can be tracked through the simple application of herbicides to growing plants or through molecular techniques. As the full sequence of the mutation lines is known including the causal mutation for herbicide resistance, molecular markers can be designed, such as single nucleotide polymorphic markers, for the selection of plants and lines carrying the resistance. These markers along with herbicide bioassays facilitate the development of at least FOP type of ACCase herbicide resistance in rice.

[00047] Selection of herbicide resistant rice in a breeding program is accomplished by spraying the progeny material with herbicide in a bioassay to observe material inheriting the resistance. Alternatively line ML0831265-01493 may be selected by sequencing the gene region containing the mutation or by creating a single nucleotide polymorphic marker to detect the mutation.

Production of Hybrid Rice Tolerant/Resistant to ACCase Inhibitor

[00048] The practical development of the trait for weed control in rice based on the application of ACCase FOP type of herbicides is now possible. Previously these herbicides had no application in rice because they killed the rice plants. Any of the rice lines described is suitable to be developed into a rice cultivar or hybrid and used in rice production as a weed control method. The resistant trait was demonstrated to be fully heritable allowing for breeding and development. The trait was demonstrated to survive and produce normal seed set after application of FOP herbicides at rates that normally kill rice. In addition the trait was amendable to application with multiple FOP herbicides. The level of herbicide resistance is such to allow complete control of red rice and other grass type weeds.

The trait is fully selectable with either an herbicide bioassay or a molecular marker allowing selection and breeding strategies to develop new rice cultivars and hybrids with FOP herbicide resistance. The resistance provided in line ML0831265-01493 is due to a single gene acting partially dominantly or fully dominantly making it ideally suited to be backcrossed into current commercial cultivars Alternatively the line ML0831265-01493, though lacking some key quality characteristics for some markets, is still agronomically suitable to be used as a parent line in a pedigree breeding program. Alternatively the line if crossed with certain female lines may be used to directly produce hybrid seed carrying herbicide resistance, as described herein.

Suitable lines that upon conversion with genes disclosed herein produce commercial rice resistant to ACCase inhibiting herbicides rice seeds deposited in the ATCC as PTA-8504, PTA-8505, PTA-836 and PTA-6795. Conversion may be by breeding or recombinant methods.

EXAMPLES

Example 1: Results of quizalofop herbicide rate response of ML0831265-01493 plants

[00052] Rice lines with the G2096S mutation were tested for level of resistance to quizalofop herbicide (Assure II^®) by testing a series of different application rates of the herbicide. The herbicide rate treatments were as shown in FIG. 6. All treatments were applied at the 2-3 leaf stage. The plots were evaluated twenty-one days after application. The spray was applied in a volume of 10 gal/acre and with 1 % Crop Oil Concentrate. The treatments were evaluated as the percent injury compared to an unsprayed control plot.

[00053] The source of the G2096S mutation was line ML0831265-01493. A

sample of seed from ML0831265-01493 is deposited with the ATCC. This line is like R0146 except that it has resistance to some ACCase herbicides due to a mutation causing an amino acid change to serine instead of glycine at position 2096 in the ACCase gene.

Four different selections of ML0831265-01493 all with the G2096S mutation were replicated three times in each treatment and tested along with the non-mutant R0146 line. The results are based on scoring twenty-one days after herbicide application.

Example 2: Results of ACC inhibitor herbicide rate response of rice lines with the G2096S mutation

Rice lines (ML0831265-01493) with the G2096S mutation were tested for response to different ACCase inhibiting herbicides. A set of herbicides (FIG. 7) were selected and applied to three rice lines with the G2096S mutation along with the non-mutated original variety R0146. The response of R0146 and the mutated line is shown in FIG. 7. The rate of herbicide application is twice the level of the labeled rate except for quizalofop and clethodim, which was twice the rate selected as a rate satisfactory to kill rice.

All treatments were applied at the 2-3 leaf stage about 20 days after seeds were planted. The plots were evaluated twenty-one days after application. The spray was applied in a volume of 10 gal/acre and with 1% Crop Oil Concentrate. The treatments were evaluated as the percent injury compared to an unsprayed control plot.

A sample of seed from ML0831265-01493 is deposited with the ATCC. This line is similar to R0146 except that it has resistance to some ACCase herbicides due to a mutation causing an amino acid change to serine instead of glycine at position 2096 in the ACCase gene.

Three different selections of ML0831265-01493 all with the G2096S mutation were replicated two or three times in each treatment and tested along with the non-mutant R0146 line. The results are based on scoring twenty-one days after herbicide application.

Different mutations in the ACCase encoding gene are often associated with resistance to specific types of ACCase inhibiting herbicides (FOPS), (DIMS). The specificity of different mutations thus offers the possibility of developing multiple modes of action for weed control in rice. For example, FIG. 9 shows that the G2096S mutation disclosed herein conveys greater tolerance in rice to two common FOP type of ACCase inhibiting herbicides than an alternative recently published mutation. The different response of rice line ML0831265-01493 to FOP herbicides shows that commercial development of this line or essentially similar lines provides a new mode of action for weed control not currently available from other sources.

Example 3: Mutant rice line ML0831265-01493 shows no apparent differences from non-mutated R0146 rice in characteristics other than ACCase inhibitor resistant rice.

[00060] In research plots the mutant line ML0831265-01493 was observed side by side with the original non-mutant line R0146. No observable differences were identified. The plants showed the same growth pattern. The health and robustness of the plants also appeared similar without any detectable differences. Some characteristics where measured also showed new significant differences between the mutant line ML0831265-01493 and the unmutated line R0146 (Table 2). The G2096S mutation in line ML0831265-01493 shows no negative effects on the normal growth or fitness of the plants.

Example 4: Development of DNA markers to select plants with the mutation.

[00061] DNA markers allow selection for certain traits without having to observe the phenotype. In the instance of line ML0831265-01493 the mutation causing the resistance is known including the specific DNA sequence and surrounding sequence. Knowing the sequence allows the design, making, and use of any marker system that will detect single nucleotide polymorphisms.

[00062] In most single nucleotide detection assays a primer is labeled with a

specific fluorescent dye and synthesized based on the DNA sequence to anneal with one nucleotide at the mutation site. A second primer is also made carrying a second fluorescent dye of a different color to anneal with the alternative nucleotide at the mutation site. Both primers are then allowed to anneal with a sample of DNA from an individual plant. After washing only the primers which have an annealing match remain in the sample. The fluorescence is measured and based on the color detected the nucleotide at the mutation site is determined. A single color indicates the sample was homozygous for either the non-mutant type or the mutant type, depending on the color detected, and detection of both colors indicates the sample was heterozygous. Applying single nucleotide markers for the mutation allows selection for herbicide resistance without having to observe effects of herbicide application on the plants. Testing by either molecular marker or phenotyping is required until a new line is proven to be homozygous for the mutation. Molecular markers show if a line or plant is homozygous or heterozygous allowing detection of homozygosity one generation earlier than is possible with observing the effect of applying herbicides.

Example 5: Integration of ACCase resistance into commercial lines

[00065] Plants are grown from line ML0831265-01493 with the G2096S mutation

(donor parent) and plants from the recurrent parent, which in this example is P1233. The P1233 plants are emasculated following standard crossing procedures and pollinated with pollen from plants of line ML0831265-01493. 2-4 inches of leaf material of individual plants are collectively used to make the crosses and to analyze the plants with molecular markers to identify a set of polymorphic markers. It is best to identify approximately 100 polymorphic markers evenly spaced across the rice genome. Harvest Fl seed from the P1233 plants, which was used as the female parent in the cross.

[00066] Fl seeds and the recurrent parent line, P1233 are planted and grown again.

The Fl seedlings are sprayed with herbicide to verify successful crossing occurred from the herbicide resistant donor parent. Those plants not inheriting resistance will die. In addition the plants could also be tested with a few of the polymorphic markers to verify they were true Fl plants, i.e. received markers from both parents, the crossing process is repeated by using the resistant Fl plants as the male or pollen donor parent to plants from line P1233 emasculated and used as the female parent. After seeds mature the BC1F1 seed is harvested.

[00067] The BC1F1 seeds and the recurrent parent line, P1233 are planted and grown. The BC1F1 seedlings are sprayed with herbicide to identify those that inherited the herbicide tolerance mutation from the donor parent. Leaf tissue is collected from tolerant seedlings and submitted to the lab for analysis with polymorphic markers. Five plants are selected that show the highest portion of the line P1233 genome based on the marker analysis. They are used as a pollen donor onto emasculated plants of line P1233. After seeds mature, the BC2F1 seed is harvested. 47644

[00068] The BC2F1 and the recurrent parent line, PI 233 are planted and grown.

The BC2F1 seedlings are sprayed with herbicide to identify those that inherited the herbicide tolerance mutation from the donor parent. Leaf tissue is collected from the tolerant seedlings and submitted to the lab for analysis with all unfixed (segregating) markers. Five plants are selected that show more than 95% recovery of the PI 233 genome and these are allowed to self pollinate. The backcrossing step is repeated if no individuals show at least 95% recovery of the PI 233 genome. BC2F2 seed is harvested from individual self pollinating plants.

[00069] At least 24 individual BC2F2 seeds from each plant are planted and grown.

Leaf tissue is collected; DNA is extracted and sequenced to identify individuals that carry the G2096S mutation in homozygous condition. The plants are allowed to self pollinate. The BC2F3 seed from these plants is harvested and identified as a new herbicide tolerant line of P1233. Lines or progeny rows are grown in head rows and selections for the best row are made to advance to hybrid crossing and yield trials.

[00070] The same process may also be followed to develop other lines that carry resistance to ACCase FOP type herbicides. The recurrent parent is chosen as an S- line to develop resistance in a female parent used in hybrid production. Alternatively resistance may be developed in more than one recurrent parent. One recurrent parent line is the male line used in a hybrid and the other is the female line used in the hybrid to make a hybrid that carried the resistance (G2096S mutation) in a homozygous condition. Other examples of recurrent parents may be lines carrying current commercial traits such as other herbicide resistances or even transgenic traits. Other parents could be derived from other screenings of mutant lines and selected to combine multiple traits into a single line.

Example 6: Development of new commercial lines with resistance to ACCase type FOP herbicides

[00071] Resistance in rice to ACCase FOP type herbicides is developed in either hybrid parent lines or varieties through a breeding approach. A careful analysis of the line ML0831265-01493 for inherent strengths and weakness is done to identify lines that will correct the weaknesses in line ML0831265-01493. Line ML0831265-01493 carrying the herbicide resistance as the male parent is used so that simple bioassays (spraying the plants with the herbicides and observing those that live as individuals that inherited the resistance) can be applied to verify successful crosses.

After selecting one or more appropriate parents a cross is first made to one selected line. Crosses with other parents could be made in later generations to contribute additional traits or genetic variation. In this example the development process will involve only one cross to PI 003 to improve the weak characteristics of the mutant line ML0831265-01493. Other parents are chosen from a mutant population carrying resistance to an alternative herbicide allowing multiple herbicides to be used for weed control in rice production. Parents are also selected that carry an already developed and commercialized herbicide resistance or transgenic trait.

In the first step the selected parent line PI 003 is emasculated being used as the female and considered as providing unique characteristics and that when recombined with line ML0831265-01493 will lead towards development of a new variety or parent line for hybrids. Pollen from the mutant line carrying the G2096S mutation ML0831265-01493 is used to pollinate the emasculated plants of line PI 003. The Fl seed is harvested and planted. If desired an additional cross could be made to either of the parent lines or to another parent for the purpose of introducing other characteristics and genetic variation.

Growing the Fl seed and applying herbicide is done to verify the cross was successful. The surviving plants should be true Fl and are allowed to self to produce F2 seed. The F2 seed will then be planted and again sprayed to identify plants inheriting the herbicide resistance. The plants remaining alive should be either homozygous or heterozygous for the resistant trait. If other traits are of interest they should also be evaluated at this stage for inheritance in the F2 plants. Select among the F2 plants surviving the herbicide treatment and allow them to self pollinate to produce F3 seed. Harvest F3 seed from individual plants and maintain as an individual F3 family.

The F3 families are then planted as rows and again herbicide applied to identify the F2 plants and F3 families which are homozygous for the herbicide resistance. Selections are made among the F3 families that are homozygous for the herbicide resistance for other traits and characteristics of interest. F4 seed is harvested from the selected rows. [00076] The F4 seed is used directly in yield trials to develop a new variety or test cross to select parents to produce hybrid seed for testing in yield trials. Selections are made among the lines in the yield trials for yield and other target traits and characteristics such as quality. The F4 seed should also be increased to F5 at which selections for target traits can also be made. The F5 seed should be used again in test crosses for yield trials with hybrid seeds as well as being put directly into yield trials if a variety is to be developed.

[00077] After yield trials, including multi-location and replicated testing, and full testing of the trait response, a final selection is made to identify one or a few lines to release as a coded line. These lines are then used for seed increase and release as either a new variety or a parent in a hybrid.

Example 7: Herbicide resistance deployed in hybrids

[00078] The herbicide resistance described in line ML0831265-01493 is likely either dominant or partially dominant. The resistant event is deployed in a hybrid by being integrated into the male parent, the female parent or both parents. Any combination is developed for successfully controlling weeds in rice with an ACCase FOP type of herbicide. Through following the process of the examples above parent lines are developed to carry the ACCase herbicide resistance. These parent lines are then used in a hybrid seed production system to produce hybrid seed carrying the ACCase resistance to FOP type of herbicides. The seed production process involves planting the female line in rows next to the male lines. The female lines are male sterile so as to prevent self pollination. The female lines then are pollinated by the male lines and harvested to produce Fl seed. The Fl seed is hybrid seed and is planted by growers to produce rice grain. In the situation where a variety is developed, the seed is planted in isolation and then harvested to sell to growers to produce rice grain.

[00079] In preparation to produce either hybrid or variety seed from selected lines carrying the ACCase resistance derived from ML0831265-01493 lines must first be purified, extensively tested, and increased.

[00080] In the example of producing hybrid seed with resistance to ACCase FOP type of herbicides the resistance may be provided in the male parent, the female parent or both parents. Providing the resistance in both parents could be necessary to have a high enough level of resistance to prevent herbicide damage to the rice 12 047644 when herbicide is applied. However, a more suitable delivery mechanism in hybrids is if the resistance can be provided in either only the female or only the male parent.

By providing the resistance in only the male parent offers a process to eliminate any female selfed seed in a growers field. With resistance being only provided from the male parent then when a grower applies herbicide to his field all of the female selfed plants will be susceptible to the herbicide and thus killed. The grower's field is chemically rogued and results in a pure stand of hybrid plants.

It is also possible to provide ACCase resistance to FOP herbicides through combining with other traits. For example resistance to herbicides with alternative modes of action or other traits such as insect resistance, drought tolerance.

Combining with other traits could be either by conventional or transgenic methods.

In one example ACCase resistance is integrated into the female parent and an alternative herbicide resistance is integrated into the male parent. The seed is resistant to both herbicides and a grower may use either or both herbicides for weed control. Alternatively only one type of resistance is delivered in a single hybrid. Both systems as well as other strategies all provide growers with additional options for controlling weeds and will likely extend the useful life of the herbicides. In the case of deploying in only one parent or only one hybrid any red rice that develops resistance through outcrossing will only inherit one type of resistance and will still be controllable through application of an herbicide with an alternative mode of action.

Example 8: Seed Production

The herbicide resistance may be used for production of hybrid seed. As an example, if the female parent is developed with resistance to ACCase FOP type herbicide through inheritance from line ML0831265-01493 then the herbicide could be used in seed production to eliminate the male parent. By deploying into the female parent, making it resistant, then the herbicide is applied to the seed production field to kill the male plants before setting seed but after pollination. In this way the male parent is prevented from setting seed and allows seed production fields to be harvested as a bulk instead of only harvesting the female rows. In addition the purity of hybrid seed may also be verified through deploying the resistances in only one parent. Any selfed seed of the other parent are killed by application of the herbicide.

Deposit Information

[00085] A deposit of the RiceTec, Inc. ML0831265-0149 disclosed above and recited in the appended claims has been made with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 201 10. The date of deposit was May 30, 2012. All restrictions will be removed upon granting of a patent, and the deposit is intended to meet all of the requirements of 37 C.F.R. .sctn..sctn.1.801 -1.809. The ATCC Accession Number is PTA-12933. The deposit will be maintained in the depository for a period of thirty years, or five years after the last request, or for the enforceable life of the patent, whichever is longer, and will be replaced as necessary during that period.

[00086] Seeds are being prepared from the other herbicide resistant lines for

deposit.

[00087] While a number of exemplary aspects and embodiments have been

discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.

DEFINITIONS

[00088] In the description and tables which follow, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided:

[00089] Allele. Allele is any one of many alternative forms of a gene, all of which generally relate to one trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes. [00090] Backcrossing. Process of crossing a hybrid progeny to one of the parents, for example, a first generation hybrid Fl with one of the parental genotypes of the Fl hybrid.

[00091] Blend. Physically mixing rice seeds of a rice hybrid with seeds of one, two, three, four or more of another rice hybrid, rice variety or rice inbred to produce a crop containing the characteristics of all of the rice seeds and plants in this blend.

[00092] Cell. Cell as used herein includes a plant cell, whether isolated, in tissue culture or incorporated in a plant or plant part.

[00093] Cultivar. Variety or strain persisting under cultivation.

[00094] Embryo. The embryo is the small plant contained within a mature seed.

[00095] Essentially all the physiological and morphological characteristics. A plant having essentially all the physiological and morphological characteristics of the hybrid or cultivar, except for the characteristics derived from the converted gene.

[00096] Grain Yield. Weight of grain harvested from a given area. Grain yield could also be determined indirectly by multiplying the number of panicles per area, by the number of grains per panicle, and by grain weight.

[00097] Locus. A locus is a position on a chromosome occupied by a DNA

sequence; it confers one or more traits such as, for example, male sterility, herbicide tolerance, insect resistance, disease resistance, waxy starch, modified fatty acid metabolism, modified phytic acid metabolism, modified carbohydrate metabolism and modified protein metabolism. The trait may be, for example, conferred by a naturally occurring gene introduced into the genome of the variety by backcrossing, a natural or induced mutation, or a transgene introduced through genetic transformation techniques. A locus may comprise one or more alleles integrated at a single chromosomal location.

[00098] Plant. As used herein, the term "plant" includes reference to an immature or mature whole plant, including a plant from which seed or grain or anthers have been removed. Seed or embryo that will produce the plant is also considered to be the plant.

[00099] Plant Part. As used herein, the term "plant part" (or a rice plant, or a part thereof) includes protoplasts, leaves, stems, roots, root tips, anthers, seed, grain, embryo, pollen, ovules, cotyledon, hypocotyl, glumes, panicles, flower, shoot, tissue, cells, meristematic cells and the like. [000100] Quantitative Trait Loci (QTL). Genetic loci that controls to some degree numerically measurable traits that are usually continuously distributed.

[000101] Regeneration. Regeneration refers to the development of a plant from tissue culture.

[000102] Single Gene Converted (Conversion). Single gene converted (conversion) includes plants developed by a plant breeding technique called backcrossing wherein essentially all of the desired morphological and physiological characteristics of an inbred are recovered, while retaining a single gene transferred into the inbred via crossing and backcrossing. The term can also refer to the introduction of a single gene through genetic engineering techniques known in the art.

Table 1 - Rice lines derived from the surviving plants in the permanent mutant population confirmed to carry resistance to quizalofop herbicide. The ATCC accession number is shown for the line with the mutation in the carboxyl transferase region of the ACCase gene. The ATCC accession number is pending for the other two lines.

Table 2 - Comparison of the mutant line ML0831265-0149 with the original unmutated line R0146 showing high similarity between the two lines.

Claims

CLAIM:

1. A method for controlling weeds in a rice field the method comprising:

(a) having rice in the field wherein the rice is resistant to an ACCase inhibiting herbicide due to a mutation, and wherein the rice is derived from ATCC Accession No. PTA-12933 through breeding, tissue culture, or transformation; and

(b) applying the herbicide to the field.

2. A method for controlling weeds in a rice field, the method comprising:

(a) having herbicide resistant rice in the rice field wherein the rice is resistant to a plurality of herbicides; and

(b) contacting the rice field with a plurality of the herbicides to which the rice plants are resistant.

3. The method of claim 2 wherein the plurality of herbicides comprises at least one ACCase inhibiting herbicide and resistance is due to a mutation.

4. The method of claim 2 wherein the herbicide resistant rice is PTA-12933.

5. The method of claim 1 or 3 wherein the ACCase inhibiting herbicide is a FOP herbicide.

6. The method of claim 2 wherein the rice is selected from Table 1.

7. The method of claim 1 or 3 wherein the ACCase herbicides are selected from the group consisting of quizalofop, clodinafop-propargyl, haloxyfop-methyl, and

combinations thereof.

8. The method of claim 1 or 3 wherein the mutation associated with resistance is G2096S.

9. The method of claim 4 wherein the source of the mutation is seed deposited under ATCC Accession No. PTA-12933.

10. A method for developing rice with resistance to ACCase inhibiting herbicides, wherein the resistance to ACCase inhibiting herbicides is derived from the genes of ATCC Accession No. PTA-12933, the method comprising:

(a) introgressing rice with ATCC Accession No. PTA-12933 into a non resistant source of rice; and

(b) confirming the rice is resistant.

1 1. A method of producing an herbicide resistant rice plant wherein the method comprises transforming a rice plant with a transgene and wherein the transgene confers resistance to an herbicide selected from the group of claim 7.

12. The method of claim 1 1 wherein the transgene has a nucleic acid sequence as in SEQ ID NO: 1.

13. The method of producing herbicide resistant rice of claim 1 1, the method

comprising transforming rice with a nucleic acid molecule having SEQ ID NO: 1 in any association of promoters, terminators, selectable markers, or other domains of the ACCase coding gene.

14. A method for producing a rice seed, wherein the method comprises crossing the plant of claim 15, 17, 19, 22 or 23 with a different rice plant and harvesting the resultant hybrid rice seed.

15. An herbicide resistant rice plant produced from a seed produced by the method of claim 14.

16. A rice seed designated ML0831265-01493, wherein a representative sample of seed was deposited under ATCC Accession No. PTA- 12933.

17. A rice plant, or a part thereof, produced by growing the seed of claim 16.

18. Pollen or an ovule of the plant of claim 15 or 17.

19. A rice plant, or a part thereof, having all of the physiological and morphological characteristics of the rice plant of claim 16.

20. A tissue culture produced from protoplasts or cells from the rice plant of claim 15 or 17 wherein said cells or protoplasts of the tissue culture are produced from a plant part selected from the group consisting of leaves, pollen, embryos, cotyledon, hypocotyl, meristematic cells, roots, root tips, pistils, anthers, flowers, stems, glumes and panicles.

21. A protoplast produced from a plant of claim 15 or 17.

22. A rice plant regenerated from the tissue culture of claim 20, wherein the plant has all the morphological and physiological characteristics of rice plant of claim 16.

23. An herbicide resistant rice plant comprising a DNA sequence comprising of a plastidic carboxylase transferase domain of an ACCase coding gene which confers resistance to an ACCase herbicide selected from the group consisting of quizalofop, clodinafop-propargyl, haloxyfop-methyl, and combinations thereof.

24. The herbicide resistant rice plant according to claim 23 comprising SEQ ID NO 1.

25. An isolated DNA sequence comprising SEQ ID NO: 1 of a plastidic carboxylase transferase domain of an ACCase coding gene, which conveys tolerance to ACCase inhibiting chemicals.

26. A mutant ACCase enzyme comprising an amino acid sequence as in FIG. 5 (SEQ ID NO: 7) that confers resistance to ACCase inhibitors in rice that have the enzyme.

27. The mutant enzyme of claim 26, that conveys larger differential response to FOPs than a published response of rice with a different mutation.