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 personsare required to respond to a collection of information unlessit displays a valid OMB control number.
`PROVISIONAL APPLICATION FOR PATENT COVERSHEET- Page1 of 2
`This is a requestforfiling a PROVISIONAL APPLICATION FOR PATENTunder 37 CFR 1.53(c).
`
`Express Mail Label No.
`
`Given Name(first and middle[if any] )
`
`INVENTOR(S)
`
`Residence
`City and either State or Foreign Country)
`
`Holly Springs, North Carolina
`separately numbered sheets attached hereto.
`1
`Additional inventors are being named on the
`TITLE OF THE INVENTION(500 characters max):
`HERBICIDE-TOLERANT PLANTS
`
`Direct all correspondenceto:
`CORRESPONDENCE ADDRESS
`[x] The address corresponding to Customer Number:
`OR
`
`26158
`
`Firm or
`Individual Name
`
`Address
`
`Telephone
`ENCLOSED APPLICATION PARTS(checkall that apply)
`[_]Apptication Data Sheet. See 37 CFR 1.76[| CD(s), Number of CDs
`Drawing(s) Number of Sheets
`48
`[} Other(specify)
`[x]Specification (e.g. description ofthe invention) NumberofPages
`69
`
`,
`
`If the specification and drawings exceed 100 sheets of paper, an application size fee is also
`Fees Due: Filing Fee of $220 ($110 for small entity).
`due, which is $270 ($135 for small entity)
`for each additional 50 sheets orfraction thereof. See 35 U.S.C. 41(a)(1)(G) and 37 CFR 1.16(s).
`METHOD OF PAYMENTOF THEFILING FEE AND APPLICATIONSIZE FEE FOR THIS PROVISIONAL APPLICATION FOR PATENT
`[| Applicant claims smailentity status. See 37 CFR 1.27.
`220.00
`Accheck or money order made payable to the Director ofthe United States Patent and Trademark Office
`°TAL FEE AM
`is enclosed to coverthefiling fee and application size fee (if applicable).
`OUNT ($)
`"
`[| Paymentby credit card. Form PTO-2038is attached.
`TheDirectoris hereby authorized to chargethefiling fee and application size fee(if applicable) or credit any overpaymentto Deposit
`Account Number:
`09-0528
`
`USE ONLYFORFILING A PROVISIONAL APPLICATION FOR PATENT
`
`PGR2021-00114 Ex. 1014
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`Page 001
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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 respond to a collection of information unlessit displays a valid OMB contro! number.
`
`
`
`
`The invention was made by an agency of the United States Governmentor under a contract with an agencyof the United States Government.
`[x] No.
`[| Yes, the nameof the U.S. Government agency and the Government contract 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 USPTOto 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 (unless 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
`
`.
`
`Date
`
`July 16, 2010
`
`TYPED or PRINTED NAME
`
`Lawrence J. Carroll
`
`REGISTRATION NO.
`(if appropriate)
`
`40,940
`
`TELEPHONE
`
`(202) 467-6900
`
`Docket Number:
`
`B248 1010.P2
`
`WCSR 4417916v1
`
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`SUPPLEMENTAL SHEET TO
`PROVISIONAL APPLICATION COVER SHEET(PTO/SB/16)
`(Page 1 of1)
`
`
`
`Scots L. Mankin
`
`Docket Number
`
`B248 1010.P1
`
`
`
`
`
`
`
`
`City and either State or Foreign Count
`
`Raleigh, North Carolina
`Whitt
`
`
`
`Cary, North Carolina
`Stevenson-Paulik
`
`Apex, North Carolina
`Carlson
`
`
`
`
`Given Name(first and middle [if an
`
`Family or Surname
`
`WCSR 4417916v1
`
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`B248 1010.P2
`
`HERBICIDE-TOLERANT PLANTS
`
`BACKGROUNDOFTHE INVENTION
`
`Rice is one of the most important food crops in the world, particularly in Asia.
`[0001]
`Rice is a cereal grain produced by plants in the genus Oryza. The two mostfrequently
`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 ofthese 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 ofthe rice crop. Since red
`tice belongs to the samespeciesas cultivated rice (Oryza sativa), their genetic makeupis
`very similar. This genetic similarity has made herbicidal controlof red rice difficult.
`
`Domestic rice tolerant to imidazolinone herbicides have been developed and are
`[0002]
`currently marketed under the tradename CLEARFIELD®. Imidazolinone herbicides inhibit 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.
`
`[0003]
`
`Acetyl-CoenzymeA carboxylase (acetyl-Coenzyme A carboxylase; EC 6.4.1.2)
`
`enzymes synthesize malonyl-CoAas thestart 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 homomericprotein, likely a homodimer.
`
`[0004]
`
`ACCase enzymesin 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.
`
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`B248 1010.P2
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`A number of ACCase-inhibitor-tolerance (AIT) mutations have been found in
`[0005]
`monocot weedspecies exhibiting tolerance toward one or more DIM or FOPherbicides.
`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 catalyticsite.
`
`DIMsand FOPsare important herbicides and it would be advantageousifrice
`[0006]
`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 severe fitness penalty in the tolerant plant. Therefore,
`there remainsa needin the art for an AIT rice that also exhibits nofitness penalty. This need
`
`and others are met by the present invention.
`
`BRIEF SUMMARYOF THE INVENTION
`
`The present inventionrelates to herbicide-tolerant plants and methods of
`[0007]
`producingand treating herbicide-tolerant plants. In one embodiment, the present invention
`providesa 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 of a rice
`plant. Typically, an herbicide-tolerantrice plant of the invention expressesan acetyl-
`Coenzyme A carboxylase (ACCase) in whichthe 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 aminoacid residues are typically referred to
`
`in referenceto their position in the Alopecurus myosuroides (blackgrass) ACCase sequence
`
`(Genbank CAC84161.1) and denoted with an (Am). Examples of amino acid positionsat
`
`which an acetyl-Coenzyme A carboxylase of a 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 more of 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
`
`aminoacid at position 1,781(Am) is other than isoleucine; the aminoacid at position
`
`1,999(Am)is other than tryptophan; the amino acid at position 2,027(Am)is other than
`
`tryptophan; the aminoacid at position 2,041(Am)is other than isoleucine; the amino acid at
`
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`B248 1010.P2
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`position 2,078(Am)is other than aspartate; the amino acid at position 2088(4m)is other than
`cysteine; or the aminoacidat 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 more ofthe
`following: the aminoacid at position 1,781(4m) is leucine or alanine; the aminoacid 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 aminoacid 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 provides methods of producing herbicide-tolerant
`[0008]
`plants and plants produced by such methods. An example of a plant produced by the
`methodsofthe invention is an herbicide-tolerantrice plant whichis tolerant to at least one
`herbicide that inhibits acetyl-Coenzyme A carboxylase activity at levels of herbicide that
`would normally inhibit the growth of said plant, wherein the herbicide-tolerant plantis
`produced by: a) obtaining cells from a plant that 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.
`
`[0009]
`
`The present invention provides plants expressing acetyl-Coenzyme A carboxylase
`
`enzymes comprising defined amino acid sequences. For example, the present invention
`
`provides a rice plant, wherein one or more of the genomesofsaid rice plant encodea protein
`
`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 amino acid at position 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 amino acid at position 2,096(Am)is alanine. Table 3
`
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`B248 1010.P2
`
`below provides an alignmentof the Alopecurus myosuroides acetyl-Coenzyme A carboxylase
`sequence (SEQ ID NO:1), the Oryzasativa 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 sequencesof the invention indicated.
`
`In another embodiment, the present invention comprises seeds deposited in an
`[0010]
`acceptable depository in accordancewith the BudapestTreaty, cells derived from suchseeds,
`plants grown from such seedsandcells derived from such plants, progeny ofplants grown
`from such seed andcells derived from such progeny. The growth of plants produced from
`
`deposited seed and progenyofsuchplantswill typically be tolerant to acetyl-Coenzyme A
`carboxylase-inhibiting herbicidesat levels of herbicide that would normally inhibit the
`growth of a corresponding wild-type plant. In one embodiment, the present invention
`provides a 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 ATCC accession number PTA-10267 as
`
`well as any progeny of the plant grown from a seed having ATCC accession number PTA-
`
`10267 so long as such plants or progeny have the herbicide tolerance characteristics of the
`
`plant grown from a seed having ATCCaccession number PTA-10267. The present invention
`
`also encompassescells cultured from such seeds and plants and their progeny produced from
`
`the cultured cells.
`
`[0011]
`
`An herbicide-tolerant plant of the invention may be a memberofthe species O.
`
`sativa. Herbicide-tolerant plants of the invention are typically tolerantto
`
`aryloxyphenoxypropionate herbicides, cyclohexanedione herbicides, phenylpyrazoline
`
`herbicides or combinations thereof at levels of herbicide that would normally inhibit the
`
`growth of a corresponding wild-type plant, 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-tolerant plant that is mutagenized, for example, a mutagenized rice
`
`plant. The present invention also encompassescells derived from the plants and seeds of the
`
`herbicide-tolerant plants described above.
`
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`B248 1010.P2
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`The present invention provides methods for controlling growth of weeds. In one
`[0012]
`embodiment, the present invention provides a methodof controlling growth of weedsin
`vicinity to rice plants. Such methods may comprise applying to the weeds andrice plants an
`amountof an acetyl-Coenzyme A carboxylase-inhibiting herbicide that inhibits naturally
`occurring acetyl-Coenzyme A carboxylase activity, wherein said rice plants comprise altered
`acetyl-Coenzyme A carboxylase activity such that said rice plants are tolerant to the applied
`amount ofherbicide. Methodsof the invention may be practiced with any herbicide that
`interferes with acetyl-Coenzyme A carboxylase activity including, but not limitedto,
`aryloxyphenoxypropionate herbicides, cyclohexanedione herbicides, phenylpyrazoline
`herbicides or combinationsthereof.
`
`The present invention provides a method for controlling growth of weedsin
`[0013]
`vicinity to rice plants. One example of such methods may comprise applying one or more
`herbicides to the weedsandto the rice plants at levels of herbicide that would normally
`inhibit the growth ofa rice plant, wherein at least one herbicide inhibits acetyl-Coenzyme A
`carboxylaseactivity. Such methods maybepracticed with any herbicide that inhibits acetyl-
`Coenzyme A carboxylase activity. Suitable examples of herbicides that may beused in the
`practice of methodsof controlling weeds include, butare not limited to,
`aryloxyphenoxypropionate herbicides, cyclohexanedioneherbicides, phenylpyrazoline
`
`herbicides or combinationsthereof.
`
`The present invention encompasses a methodfor controlling growth of weeds.
`[0014]
`Oneexample 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; and (c) applying one or more acetyl-Coenzyme A carboxylase-inhibiting
`
`herbicides to the hybrid rice and to the weedsin vicinity to the hybridrice 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 used in the practice of methods of controlling weeds
`
`include, but are not limited to, aryloxyphenoxypropionate herbicides, cyclohexanedione
`
`herbicides, phenylpyrazoline herbicides or combinationsthereof.
`
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`B248 1010.P2
`
`In another embodiment, the present invention includes a methodfor selecting
`[0015]
`herbicide-tolerant rice plants. One example of such methods may comprise(a) crossing an
`herbicide-tolerantrice plant with other rice germplasm,and harvesting the resulting hybrid
`rice seed; (b) planting the hybrid rice seed; (c) applying one or more herbicidesto 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 used in the practice of methodsofcontrolling weeds include, but
`are not limited to, aryloxyphenoxypropionate herbicides, cyclohexanedioneherbicides,
`phenylpyrazoline herbicides or combinations thereof.
`
`The present invention also encompasses a method for growing herbicide-tolerant
`[0016]
`tice plants. One example of such a method comprises (a) planting rice seeds; (b) allowing
`the rice seeds to sprout; (c) applying one or moreherbicidesto the rice sprouts at levels of
`herbicide that would normally inhibit the growth of a rice plant, whereinat least one ofthe
`herbicides inhibits acetyl-Coenzyme A carboxylase. Such methods may be practiced 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, cyclohexanedioneherbicides,
`
`phenylpyrazoline herbicides or combinationsthereof.
`
`In one embodiment, the present invention provides a seed of an herbicide-tolerant
`[0017]
`rice plant. Such seed maybe used to grow herbicide-tolerant rice plants, wherein a plant
`
`grown from the seedis 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. Examplesof herbicides to which plants grown from seeds of the invention would be
`
`tolerant include but are not limited to, aryloxyphenoxypropionate herbicides,
`
`cyclohexanedione herbicides, phenylpyrazoline herbicides or combinations thereof.
`
`[0018]
`
`In another embodiment, the present invention provides a seedofa rice plant,
`
`wherein a plant grown from the seed expresses an acetyl-Coenzyme A carboxylase (ACCase)
`
`in which the amino acid sequence differs from an amino acid sequenceof an acetyl-
`
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`B248 1010.P2
`
`Coenzyme A carboxylase of a wild-typerice plant at one or moreof the followingpositions:
`1,781(Am), 1,999(Am), 2,027(Am), 2,041(Am), 2,078(Am), 2088(Am)or 2,096(Am).
`Examplesof differences at these aminoacid positions include, but are not limited to, one or
`more ofthe 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 aminoacidat position
`2,027(Am)is other than tryptophan; the aminoacidat position 2,041(Am)is other than
`isoleucine; the aminoacid at position 2,078(Am) is other than aspartate; the aminoacid at
`position 2088(Am) is other than cysteine; or the amino acid at position 2,096(4m)is other
`than glycine. In some embodiments,a plant grown from a seed of the invention may
`expresses an acetyl-Coenzyme A carboxylase enzyme comprising an aminoacid sequence
`that comprises one or more of the following: the amino acid at position 1,781(4m)is leucine
`or alanine; the amino acid 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 amino acid at
`position 2,078(Am)is glycine; the aminoacid at position 2088(4m)is arginine or the amino
`
`acid at position 2,096(Am)is alanine.
`
`The present invention encompasses seeds of specific cultivars. One example of
`[0019]
`such seedsis a seed ofrice cultivar Indical, wherein a representative sample of seed ofsaid
`
`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 of cells 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.
`
`[0020]
`
`The present invention also provides methods for producing rice seed. Such
`
`methods may comprise crossing an herbicide-tolerant rice plant with other rice germplasm;
`
`and harvesting the resulting hybrid rice seed, wherein the herbicide-tolerantrice plant is
`
`tolerant to aryloxyphenoxypropionate herbicides, cyclohexanedione herbicides,
`
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`B248 1010.P2
`
`phenylpyrazoline herbicides or combinations thereof at levels of herbicide that would
`
`normally inhibit the growth of a rice plant.
`
`The present method also comprises methods of producing F1 hybrid rice seed.
`[0021]
`Such methods may comprise crossing an herbicide-tolerantrice plant with a different rice
`plant; and harvesting the resultant F1 hybrid rice seed, wherein the herbicide-tolerantrice
`plantis tolerant to aryloxyphenoxypropionate herbicides, cyclohexanedione herbicides,
`phenylpyrazoline herbicides or combinations thereofat levels of herbicide that would
`
`normally inhibit the growth of a rice plant.
`
`Thepresentinvention also provides methods of producing herbicide-tolerant rice
`[0022]
`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 encodes an acetyl-
`Coenzyme A carboxylase enzymethat 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 methodsofthe invention, for example, the cell may be in the form
`
`of a 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 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 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 amino acid 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-tolerant plant
`
`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 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 usedin the practice of the
`
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`Ex. 1014
`RiceTec, Inc.
`Page 011
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`PGR2021-00114
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`

`

`B248 1010.P2
`
`methodsofthe invention, for example, the cell may be in the form ofa callus. The present
`invention also encompasses herbicide-tolerant plants produced by such methods.
`
`In one embodiment, the present invention comprises methods of producing
`[0023]
`recombinantplants. An example of a method for producing a recombinantrice plant may
`comprise transforminga cell of a rice plant with a transgene, wherein the cell was produced
`from a rice plant expressing an acetyl-Coenzyme A carboxylase enzyme that confers
`tolerance to at least one herbicide is selected from the group consisting of
`aryloxyphenoxypropionateherbicides, cyclohexanedione herbicides, phenylpyrazoline
`herbicides or combinationsthereof. Any suitable cell may be used in the practice of the
`methodsofthe 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 enzymesinclude butare not limited 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.
`
`Methodsofthe invention may be 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 combinationsthereof at levels of herbicide that would
`normally inhibit the growth ofa rice plant with a plant of another rice cultivar that comprises
`the desired trait to produce progenyplants; (b) selecting one or more progenyplants that
`
`havethe desiredtrait 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 desiredtrait and herbicide tolerance; and (e)
`
`repeating steps (c) and (d) three or more times in succession to produce selected 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, malesterility, herbicide tolerance, drought
`
`PGR2021-00114 Ex. 1014
`RiceTec,Inc.
`Page 012
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`Ex. 1014
`RiceTec, Inc.
`Page 012
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`

`B248 1010.P2
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`tolerance insect resistance, modified fatty acid metabolism, modified carbohydrate
`metabolism andresistance to bacterial disease, fungal disease orviral disease. An example
`of a methodfor producinga 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 malesterility. The present invention also encompasses malesterile
`
`plants produced by such methods.
`
`The present invention provides compositions comprisingplantcells, for example,
`[0025]
`cells fromarice plant. One exampleof such a composition comprises one or morecells ofa
`rice plant; and an aqueous medium, wherein the medium comprises a compoundthat inhibits
`acetyl-Coenzyme A carboxylase activity. In some embodiments, the cells may be derived
`from a rice planttolerant to aryloxyphenoxypropionate herbicides, cyclohexanedione
`herbicides, phenylpyrazoline herbicides or combinationsthereofat levels of herbicide that
`would normally inhibit the growth ofa rice plant. Any compoundthatinhibits acetyl-
`CoenzymeA carboxylaseactivity 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
`encodinga rice acetyl-Coenzyme A carboxylase (ACCase) in which the amino acid sequence
`differs from an amino acid sequence of an acetyl-Coenzyme A carboxylase of a wild-type
`
`rice plant at one or moreofthe following positions: 1,781(4m), 1,999(4m), 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 aminoacidat
`
`position 1,781(Am)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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`PGR2021-00114 Ex. 1014
`RiceTec,Inc.
`Page 013
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`Ex. 1014
`RiceTec, Inc.
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`B248 1010.P2
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`molecule of the invention may encode an acetyl-Coenzyme A carboxylase enzyme
`comprising an amino acid sequencethat comprises one or more of the following: the amino
`acid at position 1,781(Am)is leucine or alanine; the amino acidat 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 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. 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 sequenceis modified such that the encoded protein
`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 aminoacid 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.
`
`In one embodiment, the present invention provides an herbicide-tolerant, BEP
`[0027]
`clade plant. Typically such a plant is one having increased tolerance to an ACCase-inhibitor
`(ACCI) as comparedto a wild-type variety of the plant. Such plants may be produced by a
`
`process comprising either:
`
`(J) the steps of
`(a)
`
`providing BEPcladeplant cells having a first, zero or non-zero level of
`
`ACCItolerance;
`
`(b)
`
`(c)
`(d)
`
`growing thecells 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 of ACCItolerance greater thanthefirst level of step
`
`(a); and
`
`(e)
`
`generating, from ACCI-tolerant cells of step (d), a plant having a level of
`
`ACCItolerance greater than that of a wild-type variety of the plant; or
`
`(II) the steps of
`
`(f)
`
`providinga first, herbicide-tolerant, BEP clade plant having incr