PTO/SB/16 (12-08)
`Approved for use through 06/30/2010. OMB 0651-0032
`U.S. Patent and Trademark Office; U.S. DEPARTMENT OF COMMERCE
`Underthe Paperwork Reduction Act of 1995, no personsare required to respond to a collection ofinformation unlessit displays a valid OMB control number.
`PROVISIONAL APPLICATION FOR PATENT COVER SHEET- Page 1 of 2
`This is a requestforfiling a PROVISIONAL APPLICATION FOR PATENTunder37 CFR 1.53(c).
`
`Express Mail Label No.
`
`Residence
`City and either State or Foreign Count
`
`Raleigh, North Carolina
`
`Apex, North Carolina
`
`[| Firm or
`Individual Name
`Address
`
`26158
`
`
`INVENTOR(S)
`Family Name or Surname
`Given Name(first and middle [if any] )
`
`
`
`
`Scots L.
`Mankin
`
`
`
`
`
`
`Ulrich
`Schofl
`
`
`
`
`Wenck
`Durham, North Carolina
`
` Benton
`Holly Springs, North Carolina
`
`
`
`Additional inventors are being named on the
`1
`separately numbered sheets attached hereto.
`TITLE OF THE INVENTION(500 characters max):
`
`HERBICIDE-TOLERANT PLANTS
`
`
`
`
`
`
`Direct all correspondenceto:
`CORRESPONDENCE ADDRESS
`
`
`The address corresponding to Customer Number:
`OR
`
`
`
`O1Oolsc|<S|.<<
`State
`
`Telephone
`ENCLOSED APPLICATION PARTS(checkall that apply)
`
`
`[ Application Data Sheet. See 37 CFR 1.76
`[__] eD(s), Numberof CDs
`Drawing(s) Number of Sheets
`[| Other(specify)
`48
`
`
`Specification (e.g. description of the invention) Number of Pages
`217
`If the specification and drawings exceed 100 sheets of paper, an application sizefee is also
`Fees Due: Filing Fee of $220 ($110 for small entity).
`
`due, whichis $270 ($135 for small entity)
`for each additional 50 sheetsorfraction thereof. See 35 U.S.C. 41(a)(1)(G) and 37 CFR 1.16(s).
`
`METHOD OF PAYMENTOF THE FILING FEE AND APPLICATION SIZE FEE FOR THIS PROVISIONAL APPLICATION FOR PATENT
`
`
`
`[| Applicant claims smaii entity status. See 37 CFR 1.27.
`
`A check or money order made payable to the Director ofthe United States Patent and Trademark Office
`760.00
`is enclosed to coverthefiling fee and application size fee (if applicable).
`TOTAL FEE AMOUNT
`
`[| Paymentby credit card. Form PTO-2038is attached. ° @)
`
`09-0528
`TheDirector is hereby authorized to chargethefiling fee and application size fee (if applicable) or credit any overpayment to Deposit
`Account Number:
`
`
`Zip
`
`
`
`
`
`
`
`
`
`
`
`
`USE ONLYFORFILING A PROVISIONAL APPLICATION FOR PATENT
`
`PGR2021-00114 Ex. 1015
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`PROVISIONAL APPLICATION COVER SHEET
`Page 2 of 2
`
`PTO/SB/16 (12-08)
`Approvedfor use through 06/30/2010. OMB 0651-0032
`U.S. Patent and Trademark Office; U.S. DEPARTMENT OF COMMERCE
`Underthe Paperwork Reduction Act of 1995, no persons are required to respondto a collection ofinformation unlessit displays a valid OMB control number.
`
`The invention was made by an agencyof the United States Governmentor undera contractwith an agencyof the United States Government.
`No.
`Yes, the nameof the U.S. Government agency and the Governmentcontract numberare:
`
`
`
`:[|
`
`WARNING:
`Petitioner/applicant is cautioned to avoid submitting personal
`information in documents filed in a patent application that may
`contribute to identity theft. Personal information such as social security numbers, bank account numbers, or credit card
`numbers (other than a check or credit card authorization form PTO-2038 submitted for payment purposes) is never required by
`the USPTO to support a petition or an application. If this type of personal information is included in documents submitted to
`the USPTO, petitioners/applicants should consider redacting such personal information from the documents before submitting
`them to the USPTO. Petitioner/applicant is advised that the record of a patent application is available to the public after
`publication of the application (uniess a non-publication request in compliance with 37 CFR 1.213(a) is made in the application)
`or issuance of a patent. Furthermore, the record from an abandoned application may also be available to the public if the
`application is referenced in a published application or an issued patent (see 37 CFR 1.14). Checks and credit card
`authorization forms PTO-2038 submitted for payment purposes are not retained in the application file and therefore are not
`
`publicly available. SIGNATURE
`
`TYPED or PRINTED NAME
`
`Lawrence J. Carroll
`
`
`
`Date
`
`September 1, 2009
`
`REGISTRATION NO.
`(if appropriate)
`
`40,940
`
`TELEPHONE
`
`202) 467-6900
`
`Docket Number:
`
`B248 1010.P1
`
`WCSR 4196862v1
`
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`SUPPLEMENTAL SHEET TO
`PROVISIONAL APPLICATION COVER SHEET(PTO/SB/16)
`(Page 1 of 1)
`
`
`
`First Named Inventor|Scots L. Mankin Docket Number B248 1010.P1
`
`
`
`Apex, North Carolina
`
`INVENTOR(S)
`
`[eoemcinsnna|ramos|chaserSteafonmconen
`Given Name(first and middie[if an’
`Family or Surname
`City and either State or Foreign Count
`Whitt
`Raleigh, North Carolina
`Stevenson-Paulik
`Cary, North Carolina
`Carlson
`
`WCSR 4196862v1
`
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`B248 1010.P1
`
`HERBICIDE-TOLERANT PLANTS
`
`BACKGROUNDOF THE INVENTION
`
`[0001]
`
`Rice is one of the most important food crops in the world, particularly in Asia.
`
`Rice is a cereal grain produced byplants in the genus Oryza. The two most frequently
`
`cultivated species are Oryza sativa and Oryza glaberrima, with O. sativa being the most
`
`frequently cultivated domestic rice. In addition to the two domestic species, the genus Oryza
`contains more than 20 wild species. One of these wild species, Oryza rufipogon (“red rice”
`
`also referred to as Oryza sativa subsp. rufipogon) presents a major problem in commercial
`cultivation. Red rice produces red coated seeds. After harvest, rice seeds are milled to
`
`removetheir hull. After milling, domestic rice is white while wild red rice appears
`discolored. The presence of discolored seeds reducesthe value of the rice crop. Since red
`rice belongsto the same speciesas cultivated rice (Oryza sativa), their genetic makeupis
`very similar. This genetic similarity has made herbicidal control of red rice difficult.
`
`Domesticrice tolerant to imidazolinone herbicides have been developed and are
`[0002]
`currently marketed underthe tradename CLEARFIELD®. Imidazolinone herbicidesinhibit a
`plant's acetohydroxyacid synthase (AHAS) enzyme. Whencultivating CLEARFIELD®rice,
`it is possible to control red rice and other weeds by application of imidazolinone herbicides.
`Unfortunately, imidazolinone herbicide-tolerant red rice and weeds have developed.
`
`Acetyl-Coenzyme A carboxylase (acetyl-Coenzyme A carboxylase; EC 6.4.1.2)
`[0003]
`enzymes synthesize malonyl-CoA as the start ofthe de novofatty acid synthesis pathwayin
`plant chloroplasts. ACCasein 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 homomeric protein, likely a homodimer.
`
`[0004]
`ACCase enzymesin grasses are inhibited by three classes of herbicidal active
`ingredients. The two mostprevalent classes are aryloxyphenoxypropanoates (“FOPs”) and
`cyclohexanediones (“DIMs’”). In addition to these two classes, a third class
`
`phenylpyrazolines (“DENs”) has been described.
`
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`B248 1010.P1
`
`[0005]
`
`A numberof ACCase-inhibitor-tolerance (AIT) mutations have been found in
`
`monocot weed species exhibiting tolerance toward one or more DIM or FOP herbicides.
`
`Further, an AIT maize has been marketed by BASF. All such mutations are found in the
`
`carboxyltransferase domain of the ACCase enzyme, and these appearto be located in a
`
`substrate binding pocket, altering access to the catalytic site.
`
`[0006]
`DIMsand FOPsare important herbicides and it would be advantageousifrice
`could be provided that exhibits tolerance to these classes of herbicide. Currently, these
`classes of herbicide are of limited value in rice agriculture. In some cases, herbicide-
`
`tolerance-inducing mutations create a severefitness penalty in the tolerant plant. Therefore,
`there remainsa need in theart for an AITricethat also exhibits no fitness penalty. This need
`and others are met by the present invention.
`
`BRIEF SUMMARYOF THE INVENTION
`
`Thepresent invention relates to herbicide-tolerant plants and methods of
`[0007]
`producing andtreating herbicide-tolerant plants. In one embodiment, the present invention
`providesa rice plant tolerant to at least one herbicide that inhibits acetyl-Coenzyme A
`carboxylaseactivity at levels of herbicide that would normally inhibit the growth ofa rice
`plant. Typically, an herbicide-tolerantrice plant of the invention expressesan acetyl-
`Coenzyme A carboxylase (ACCase) in which the amino acid sequencediffers from an amino
`acid sequence of an acetyl-Coenzyme A carboxylase of a wild-type rice plant. By
`convention, mutations within monocot ACCase amino acid residuesare typically referred to
`in referenceto their position in the Alopecurus myosuroides (blackgrass) ACCase sequence
`(Genbank CAC84161.1) and denoted with an (4m). Examples of aminoacid positionsat
`which an acetyl-Coenzyme A carboxylase ofa herbicide-tolerant plant of the invention
`differs from the acetyl-Coenzyme A carboxylase of the corresponding wild-type plant
`include, but are not limited to, one or moreof the following positions: 1,781(Am), 1,999(Am),
`2,027(Am), 2,041(Am), 2,078(Am), 2088(Am) or 2,096(Am). Examplesofdifferencesat
`these aminoacid positions include, but are not limited to, one or moreof the following: the
`amino acid at position 1,781(Am)is other.than isoleucine; the amino acid at position
`1,999(Am)is other than tryptophan; the amino acid at position 2,027(Am)is other than
`tryptophan; the amino acid at position 2,041(4m) is other than isoleucine; the aminoacid at
`
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`B248 1010.P1
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`position 2,078(Am)is other than aspartate; the aminoacidat position 2088(4m) is other than
`
`cysteine; or the aminoacid at position 2,096(Am)is other than glycine. In some
`embodiments, the present invention providesa rice plant expressing an acetyl-Coenzyme A
`carboxylase enzyme comprising an amino acid sequence that comprises one or moreofthe
`following: the aminoacid at position 1,781(Am)is leucine or alanine; the amino acid at
`
`position 1,999(Am)is cysteine; the aminoacid at position 2,027(Am) is cysteine; the amino
`acid at position 2,041(Am) is asparagine; the aminoacidat position 2,078(Am) is glycine; the
`amino acidat position 2088(Am) is arginine or the amino acidat position 2,096(Am)is
`alanine.
`
`Thepresent invention also provides methods of producing herbicide-tolerant
`[0008]
`plants and plants produced by such methods. An exampleofa plant produced by the
`methodsof the invention is an herbicide-tolerant rice plant which is tolerant to at least one
`herbicide that inhibits acetyl-Coenzyme A carboxylaseactivity at levels of herbicide that
`would normally inhibit the growth ofsaid plant, wherein the herbicide-tolerantplantis
`produced by: a) obtainingcells from a plantthat is not tolerant to the herbicide;b)
`contacting the cells with a medium comprising one or more acetyl-Coenzyme A carboxylase
`inhibitors; and c) generating an herbicide-tolerant plant from the cells. Herbicide-tolerant
`plants produced by methodsofthe invention include, but are not limited to, herbicide-
`tolerant plants generated by performing a), b) and c) above and progeny ofa plant generated
`by performing a), b), and c) above. In one embodiment,cells used to practice methods of
`this type will be in the form ofa callus.
`
`Thepresent invention provides plants expressing acetyl-Coenzyme A carboxylase
`[0009]
`enzymes comprising defined amino acid sequences. For example, the present invention
`providesa rice plant, wherein one or more of the genomesofsaid rice plant encode a protein
`comprising a modified version of one or both of SEQ ID NOs:2 and 3, wherein the sequence
`is modified suchthat the encoded protein comprises oneor moreof the following: the amino
`acid at position 1,781(Am) is leucine or alanine; the aminoacid atposition 1,999(Am)is
`cysteine;the aminoacid at position 2,027(Am)is cysteine; the aminoacid at position
`2,041(Am) is asparagine; the amino acid at position 2,078(Am) is glycine; the aminoacid at
`position 2088(Am)is arginine or the aminoacid at position 2,096(Am)is alanine. Table 3
`
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`B248 1010.P1
`
`below provides an alignmentof the Alopecurus myosuroides acetyl-Coenzyme A carboxylase
`sequence (SEQ ID NO:1), the Oryza sativa Indical acetyl-Coenzyme A carboxylase
`sequence (SEQ ID NO:2)and the Oryza sativa Japonica acetyl-Coenzyme A carboxylase
`sequence (SEQ ID NO:3) with examples of positions where the wild type sequences may
`differ with sequences of the invention indicated.
`
`In another embodiment, the present invention comprises seeds deposited in an
`[0010]
`acceptable depository in accordance with the Budapest Treaty, cells derived from such seeds,
`plants grown from such seedsandcells derived from such plants, progeny of plants grown
`from such seed and cells derived from such progeny. The growth ofplants produced from
`deposited seed and progeny ofsuch plants will typically be tolerant to acetyl-Coenzyme A
`carboxylase-inhibiting herbicides at levels of herbicide that would normally inhibit the
`growth of a corresponding wild-type plant. In one embodiment, the present invention
`providesa rice plant grown from a seed having ATCC accession number PTA-10267. The
`present invention also encompasses mutants, recombinants, and/or genetically engineered
`derivatives of the plant grown from a seed having ATCCaccession number PTA-10267as
`well as any progenyof the plant grown from a seed having ATCCaccession number PTA-
`10267 so long as such plants or progeny havethe herbicide tolerance characteristics of the
`plant grown from a seed having ATCC accession number PTA-10267. The present invention
`also encompassescells cultured from such seeds and plants and their progeny produced from
`the cultured cells.
`
`Anherbicide-tolerant plant ofthe invention may be a memberofthe species O.
`[0011]
`sativa. Herbicide-tolerant plants of the invention are typically tolerant to
`aryloxyphenoxypropionate herbicides, cyclohexanedioneherbicides, phenylpyrazoline
`herbicides or combinations thereofat levels of herbicide that would normally inhibit the
`growth of a corresponding wild-typeplant, for example,a rice plant. In some embodiments,
`an herbicide-tolerant plant of the invention is not a GMO-plant. The present invention also
`provides an herbicide-tolerantplant that is mutagenized, for example, a mutagenized rice
`plant. The present invention also encompassescells derived from the plants and seedsofthe
`herbicide-tolerant plants described above.
`
`|
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`B248 1010.Pi
`
`The present invention provides methods for controlling growth of weeds. In one
`[0012]
`embodiment, the present invention provides a method of controlling growth of weedsin
`vicinity to rice plants. Such methods may comprise applying to the weedsandrice plants an
`amount of an acetyl-Coenzyme A carboxylase-inhibiting herbicidethat inhibits naturally
`occurring acetyl-Coenzyme A carboxylaseactivity, wherein said rice plants comprise altered
`acetyl-Coenzyme A carboxylaseactivity such that said rice plants are tolerant to the applied
`amount ofherbicide. Methodsofthe invention may be practiced with any herbicidethat
`interferes with acetyl-Coenzyme A carboxylaseactivity including, but not limited to,
`aryloxyphenoxypropionateherbicides, cyclohexanedioneherbicides, phenylpyrazoline
`herbicides or combinationsthereof.
`
`The present invention provides a method for controlling growth of weeds in
`[0013]
`vicinity to rice plants. One example of such methods may comprise applying one or more
`herbicides to the weeds andto therice plants at levels of herbicide that would normally
`inhibit the growth ofa rice plant, wherein at least one herbicide inhibits acetyl-Coenzyme A
`carboxylase activity. Such methods maybe practiced with any herbicide that inhibits acetyl-
`Coenzyme A carboxylase activity. Suitable examplesof herbicides that may be used in the
`practice of methods of controlling weeds include, butare notlimited to,
`aryloxyphenoxypropionate herbicides, cyclohexanedioneherbicides, phenylpyrazoline
`herbicides or combinations thereof.
`
`The present invention encompasses a method for controlling growth of weeds.
`[0014]
`One example of such methods may comprise (a) crossing an herbicide-tolerant rice plant
`with otherrice germplasm, and harvesting the resulting hybrid rice seed; (b) planting the
`hybrid rice seed; and (c) applying one or more acetyl-CoenzymeA carboxylase-inhibiting
`herbicidesto the hybrid rice and to the weedsin vicinity to the hybrid rice at levels of
`herbicide that would normally inhibit the growth of a rice plant. Such methods may be
`practiced with any herbicide that inhibits acetyl-Coenzyme A carboxylase activity. Suitable
`examples of herbicides that may be usedin the practice of methods of controlling weeds
`include, but are not limited to, aryloxyphenoxypropionate herbicides, cyclohexanedione
`herbicides, phenylpyrazoline herbicides or combinations thereof.
`
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`B248 1010.P1
`
`[0015]
`
`In another embodiment, the present invention includes a methodfor selecting
`
`herbicide-tolerant rice plants. One example of such methods may comprise(a) crossing an
`
`herbicide-tolerant rice plant with other rice germplasm,and harvesting the resulting hybrid
`
`rice seed; (b) planting the hybrid rice seed; (c) applying one or more herbicides to the hybrid
`
`rice at levels of herbicide that would normally inhibit the growth of a rice plant, wherein at
`least one of the herbicides inhibits acetyl-Coenzyme A carboxylase; and (d) harvesting seeds
`from the rice plants to which herbicide has been applied. Such methods maybepracticed
`with any herbicide that inhibits acetyl-Coenzyme A carboxylaseactivity. Suitable examples
`of herbicides that may be usedin the practice of methods of controlling weeds include, but
`are not limited to, aryloxyphenoxypropionate herbicides, cyclohexanedione herbicides,
`
`phenylpyrazoline herbicides or combinationsthereof.
`
`The present invention also encompasses a method for growing herbicide-tolerant
`[0016]
`rice plants. One example of such a method comprises(a) planting rice seeds; (b) allowing
`the rice seeds to sprout; (c) applying one or moreherbicides to the rice sprouts at levels of
`herbicide that would normally inhibit the growth of a rice plant, wherein at least one of the
`herbicides inhibits acetyl-Coenzyme A carboxylase. Such methods maybe practiced with
`any herbicide that inhibits acetyl-Coenzyme A carboxylase activity. Suitable examples of
`herbicides that may be used in the practice of methodsofcontrolling weeds include, but are
`not limited to, aryloxyphenoxypropionate herbicides, cyclohexanedioneherbicides,
`phenylpyrazoline herbicides or combinationsthereof.
`
`In one embodiment, the present invention provides a seed of an herbicide-tolerant
`[0017]
`rice plant. Such seed maybeusedto grow herbicide-tolerantrice plants, wherein a plant
`grown from the seed is tolerant to at least one herbicide that inhibits acetyl-Coenzyme A
`carboxylaseactivity at levels of herbicide that would normally inhibit the growth ofa rice
`plant. Examples of herbicides to which plants grown from seeds of the invention would be
`tolerant include but are not limited to, aryloxyphenoxypropionate herbicides,
`cyclohexanedioneherbicides, phenylpyrazoline herbicides or combinationsthereof.
`
`In another embodiment, the present invention provides a seed ofa rice plant,
`[0018]
`wherein a plant grown from the seed expresses an acetyl-Coenzyme A carboxylase (ACCase)
`in which the amino acid sequencediffers from an amino acid sequence ofan acetyl-
`
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`B248 1010.P1
`
`CoenzymeA carboxylase of a wild-type rice plant at one or moreof the following positions:
`
`1,781(Am), 1,999(Am), 2,027(Am), 2,041(Am), 2,078(Am), 2088(Am) or 2,096(Am).
`
`Examples of differences at these amino acid positions include, but are not limited to, one or
`
`more of the following: the amino acid at position 1,781(Am)is other than isoleucine; the
`
`aminoacid at position 1,999(4m)is other than tryptophan; the aminoacid at position
`
`2,027(Am)is other than tryptophan; the aminoacid at position 2,041(Am) is other than
`
`isoleucine; the aminoacid at position 2,078(Am)is other than aspartate; the amino acidat
`
`position 2088(4m)is other than cysteine; or the amino acid at position 2,096(Am)is other
`
`than glycine. In some embodiments,a plant grown fromaseed ofthe invention may
`
`expresses an acetyl-Coenzyme A carboxylase enzyme comprising an amino acid sequence
`
`that comprises one or moreofthe following: the aminoacid at position 1,781(Am)is leucine
`
`or alanine; the aminoacid at position 1,999(Am)is cysteine; the amino acid at position
`
`2,027(Am)is cysteine; the amino acid at position 2,041(Am) is asparagine; the aminoacid at
`
`position 2,078(Am)is glycine; the amino acid at position 2088(Am)is arginine or the amino
`
`acid at position 2,096(Am)is alanine.
`
`..
`
`[0019]
`
`The present invention encompasses seedsofspecific cultivars. One example of
`
`such seedsis a seed ofrice cultivar Indical, wherein a representative sample of seed of said
`cultivar was deposited under ATCC Accession No. PTA-10267. The present invention also
`
`encompassesa rice plant, or a part thereof, produced by growing the seeds as well as a tissue
`culture ofcells produced from the seed. Tissue cultures of cells may be produced from a
`seed directly or from a part of a plant grown from a seed, for example, from the leaves,
`pollen, embryos, cotyledons, hypocotyls, meristematic cells, roots, root tips, pistils, anthers,
`flowers and/or stems. The present invention also includes plants and their progeny that have
`been generated from tissue cultures of cells. Such plants will typically have all the
`morphological and physiological characteristics of cultivar Indical.
`
`The present invention also provides methods for producing rice seed. Such
`[0020]
`methods may comprise crossing an herbicide-tolerantrice plant with other rice germplasm;
`and harvesting the resulting hybrid rice seed, wherein the herbicide-tolerantrice plantis
`tolerant to aryloxyphenoxypropionate herbicides, cyclohexanedioneherbicides,
`
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`B248 1010.P1
`
`phenylpyrazoline herbicides or combinations thereofat levels of herbicide that would
`
`normally inhibit the growth ofa rice plant.
`
`The present method also comprises methods ofproducing F1 hybrid rice seed.
`[0021]
`Such methods may comprise crossing an herbicide-tolerant rice plant with a different rice
`plant; and harvesting the resultant F1 hybrid rice seed,wherein the herbicide-tolerant rice
`
`plant is tolerant to aryloxyphenoxypropionate herbicides, cyclohexanedioneherbicides,
`
`phenylpyrazoline herbicides or combinationsthereof at levels of herbicide that would
`
`normally inhibit the growth of a rice plant.
`
`[0022]
`
`The present invention also provides methods of producing herbicide-tolerant rice
`
`plants that may also comprise a transgene. One example of such a method may comprise
`
`transforminga cell of a rice plant with a transgene, wherein the transgene encodesan acetyl-
`
`Coenzyme A carboxylase enzyme that confers tolerance to at least one herbicide is selected
`
`from the group consisting of aryloxyphenoxypropionate herbicides, cyclohexanedione
`herbicides, phenylpyrazoline herbicides or combinations thereof. Any suitable cell may be
`used in the practice of the methods of the invention, for example, the cell may be in the form
`ofa callus. In some embodiments,the transgene may comprise a nucleic acid sequence
`
`encoding an amino acid sequence comprising a modified version of one or both of SEQ ID
`
`NOs: 2 and 3, wherein the sequence is modified such that the encoded protein comprises one
`
`or more of the following: the amino acid at position 1,781(Am) is leucine or alanine; the
`
`aminoacid at position 1,999(Am) is cysteine; the amino acid at position 2,027(Am)is
`
`cysteine; the amino acid at position 2,041(Am) is asparagine; the amino acid at position
`
`2,078(Am)is glycine; the aminoacid at position 2088(Am)is arginine or the aminoacid at
`
`position 2,096(Am)is alanine. The present invention also encompassesplants produced by
`
`such methods. Another example of a method of producing an herbicide-tolerantplant
`
`comprising a transgene may comprise transforminga cell of a rice plant with a transgene
`
`encoding an enzymethat confers herbicide tolerance, wherein the cell was produced from a
`
`rice plant or seed thereof expressing an acetyl-Coenzyme A carboxylase enzymethat confers
`
`tolerance to at least one herbicide is selected from the group consisting of
`
`aryloxyphenoxypropionate herbicides, cyclohexanedioneherbicides, phenylpyrazoline
`
`herbicides or combinations thereof. Any suitable cell may be usedin the practice ofthe
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`methodsof the invention, for example, the cell may be in the form of a callus. The present
`
`invention also encompassesherbicide-tolerant plants produced by such methods.
`
`[0023]
`
`In one embodiment, the present invention comprises methods of producing
`
`recombinant plants. An example of a method for producing a recombinantrice plant may
`
`comprise transformingacell of a rice plant with a transgene, wherein the cell was produced
`
`from a rice plant expressing an acetyl-Coenzyme A carboxylase enzymethat confers
`
`tolerance to at least one herbicide is selected from the group consisting of
`
`aryloxyphenoxypropionate herbicides, cyclohexanedioneherbicides, phenylpyrazoline
`
`herbicides or combinations thereof. Any suitable cell may be used in the practice of the
`
`methodsof the invention, for example, the cell may be in the form of a callus. A transgene
`for use in the methodsofthe invention may comprise any desired nucleic acid sequence, for
`
`example, the transgene may encode a protein. In one example, the transgene may encode an
`
`enzyme, for example, an enzyme that modifies fatty acid metabolism and/or carbohydrate
`metabolism. Examples of suitable enzymes include but are notlimited to,
`
`fructosyltransferase, levansucrase, alpha-amylase, invertase and starch branching enzymeor
`encoding an antisense of stearyl-ACP desaturase. The present invention also encompasses
`recombinant plants produced by methodsofthe invention.
`
`Methodsof the invention maybe used to producea plant, e.g., a rice plant, having
`[0024]
`any desired traits. An example of such a method may comprise:(a) crossing a rice plant that
`is tolerant to aryloxyphenoxypropionate herbicides, cyclohexanedione herbicides,
`phenylpyrazoline herbicides or combinationsthereofat levels of herbicide that would
`
`normally inhibit the growth of a rice plant with a plant of anotherrice cultivar that comprises
`the desired trait to produce progenyplants; (b) selecting one or more progenyplants that
`havethe desired trait to produce selected progeny plants; (c) crossing the selected progeny
`plants with the herbicide-tolerant plants to produce backcross progenyplants; (d) selecting
`for backcross progenyplants that have the desired trait and herbicide tolerance; and (e)
`repeating steps (c) and (d) three or moretimes in succession to produceselected fourth or
`higher backcross progeny plants that comprise the desired trait and herbicide tolerance. Any
`desired trait may be introduced using the methodsof the invention. Examplesoftraits that
`maybedesired include, butare not limited to, male sterility, herbicide tolerance, drought
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`B248 1010.P1
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`toleranceinsect resistance, modified fatty acid metabolism, modified carbohydrate
`metabolism and resistance to bacterial disease, fungal disease or viral disease. An example
`of a method for producing a malesterile rice plant may comprise transforminga rice plant
`tolerant to at least one herbicide that inhibits acetyl-Coenzyme A carboxylase activity at
`
`levels of herbicide that would normally inhibit the growth ofa rice plant with a nucleic acid
`
`molecule that confers male sterility. The present invention also encompasses malesterile
`
`plants produced by such methods.
`
`The present invention provides compositions comprising plant cells, forexample,
`[0025]
`cells from a rice plant. One example of such a composition comprises one or morecells of a
`rice plant; and an aqueous medium, wherein the medium comprises a compoundthat inhibits
`
`acetyl-CoenzymeA carboxylaseactivity. In some embodiments, the cells may be derived
`from a rice plant tolerant to aryloxyphenoxypropionate herbicides, cyclohexanedione
`herbicides, phenylpyrazoline herbicides or combinations thereof at levels of herbicide that
`would normally inhibit the growth of atice plant. Any compoundthat inhibits acetyl-
`Coenzyme A carboxylase activity may be used in the compositionsof the invention, for
`example, one or more of aryloxyphenoxypropionate herbicides, cyclohexanedione
`herbicides, phenylpyrazoline herbicides and combinations thereof.
`
`The present invention comprises nucleic acid molecules encoding all or a portion
`[0026]
`of an acetyl-Coenzyme A carboxylase enzyme. In some embodiments,the invention
`comprises a recombinant, mutagenized, synthetic, and/or isolated nucleic acid molecule
`
`encoding a rice acetyl-Coenzyme A carboxylase (ACCase) in which the amino acid sequence
`differs from an amino acid sequenceof an acetyl-Coenzyme A carboxylase of a wild-type
`rice plant at one or moreofthe following positions: 1,781(Am), 1,999(Am), 2,027(Am),
`2,041(Am), 2,078(Am), 2088(Am) or 2,096(Am). Examples of differences at these amino acid
`positionsinclude, but are not limited to, one or more of the following: the aminoacid at
`position 1,781(4m)is other than isoleucine; the aminoacid at position 1,999(Am)is other
`than tryptophan; the aminoacid at position 2,027(Am)is other than tryptophan; the amino
`acid at position 2,041(Am)is other than isoleucine; the aminoacid at position 2,078(Am) is
`other than aspartate; the aminoacidat position 2088(Am)is other than cysteine; or the amino
`acid at position 2,096(Am)is other than glycine. In some embodiments,a nucleic acid
`
`10
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`B248 1010.P1
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`molecule of the invention may encode an acetyl-Coenzyme A carboxylase enzyme
`
`comprising an amino acid sequence that comprises one or moreofthe following: the amino
`
`acid at position 1,781(Am)is leucine or alanine; the aminoacid at position 1,999(Am)is
`
`cysteine; the amino acid at position 2,027(Am)is cysteine; the aminoacid atposition
`
`2,041(Am) is asparagine; the amino acid at position 2,078(Am)is glycine; the amino acid at
`
`position 2088(Am)is arginine or the amino acid at position 2,096(Am)is alanine. In some
`
`embodiments, the invention comprises a recombinant, mutagenized, synthetic, and/or.
`
`isolated encoding a protein comprisingall or a portion of a modified version of one or both
`
`of SEQ ID NOs:2 and 3, wherein the sequence is modified such that the encodedprotein
`
`comprises one or more ofthe following: the amino acid at position 1,781(Am)is leucine or
`
`alanine; the aminoacid at position 1,999(Am)is cysteine; the aminoacidat position
`
`2,027(Am)is cysteine; the amino acid at position 2,041(Am)is asparagine; the aminoacidat
`
`position 2,078(Am)is glycine; the amino acid at position 2088(Am)is arginine or the amino’
`
`acid at position 2,096(Am)is alanine.
`
`[0027]
`
`In one embodiment, the present invention provides an herbicide-tolerant, BEP
`
`clade plant. Typically such a plant is one having increased tolerance to an ACCase-inhibitor
`
`(ACCI) as compared to a wild-type variety of the plant. Such plants may be produced by a
`
`process comprising either:
`
`(1) the steps of
`
`(a)
`
`providing BEP clade plant cells having a first, zero or non-zero level of
`
`ACCTtolerance;
`
`(b)
`
`(c)
`(d)
`
`growingthe cells in contact with a medium to form a cell culture;
`
`contacting cells of said culture with an ACCI;
`growing ACCI-contactedcells from step (c) to form a culture containing
`cells having a level o