DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 1-37 are pending.
Claims 1-37 are examined on the merits.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 28 rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 28 is rejected as indefinite for the recitation method “The method of claim 27 further comprising: (c) crossing the progeny plant with itself or another plant; and (d) repeating steps (b) and (c) for an additional 0–7 generations”. (c) step of claim 27 is“ (c) crossing the progeny plant with itself or another plant”, whereas (c) step in claim 28 is “(c) selecting a progeny plant comprising the mutation to produce a plant having reduced PPO activity”. A POSITA is not sure which (c) step to follow.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1, 3, 4, 15, 18 and 19 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by González (Matías Nicolás González et. al., Reduced Enzymatic Browning in Potato Tubers by Specific Editing of a Polyphenol Oxidase Gene via Ribonucleoprotein Complexes Delivery of the CRISPR/Cas9 System, Front Plant Sci. 2020 Jan 9:10:1649)
Claim 1 recites a modified plant, or a progeny thereof, having reduced polyphenol oxidase (PPO) activity, the plant comprising a mutation in at least one endogenous PPO1 or PPO2 gene, wherein the mutation is in a region of the endogenous PPO1 or PPO2 gene that encodes a copper binding domain of a PPO polypeptide.
Under its BRI, claim 1 encompasses any plant and its progeny having reduced polyphenol oxidase (PPO) activity that comprises a mutation in at least one endogenous PPO1 or PPO2 gene, where the mutation is located in a nucleotide region that encodes a copper-binding domain of a PPO polypeptide.
González discloses a potato plant (and regenerated plants/progeny) engineered by CRISPR/Case9 to induce mutations in the endogenous potato PPO2 gene (StPPO2), with the express purpose that editing this target gene results in lower PPO activity. They further disclose that regenerated plants contained mutations in at least one allele of StPPO2, and that these StPPO2 mutations produced lines showing reduced tuber PPO activity (p4 and fig 2). Claim 1 is anticipated by González.
Claim 3 depends from claim 1 and recites that the mutation is an insertion, deletion, or substitution.
González discloses that CRISPR/Cas9 editing produced insertions and deletions in the targeted PPO2 gene (StPPO2)(p5, table 1). Because claim 3 is written in the alternative “insertion, deletion, or substitution”, González disclosure of deletions and insertions satisfies claim 3. Accordingly, claim 3 is anticipated by González.
Claim 4 depends from claim 1 and recites that the mutation introduces a frameshift mutation, a premature stop codon, or an in-frame deletion.
González discloses that many edits in StPPO2 includes small deletions (e. g., 4-7 bp) that are predicted to cause frameshifts, and also describes larger deletions between two cut sites (including deletions that can be in-frame, depending on size/position)(p5-6, table 1). Because claim 4 is written in the alternative, the disclosure of frameshift-causing deletions is sufficient to meet claim 4. Accordingly, claim 4 is anticipated by González.
Claim 14 recites “a plant, plant cell, or seed of the modified plant of claim 1”.
González discloses editing of potato protoplasts (i.e., plant cells) by delivery of CRISPR/Cas9 ribonucleoprotein complexes, regeneration of plants, and phenotyping on tubers (i.e., a plant part)(p4 and p6). Since claim 14 is written in the alternative “plant part, plant cell, or seed”, González’s disclosure of plant cells (protoplasts) and plant parts (tubers) satisfies claim 14.
Claim 15 recites a method of reducing PPO activity comprising introducing a mutation in at least one endogenous PPO1 or PPO2 gene in a region encoding a copper binding domain.
González discloses a method of reducing PPO activity by introducing mutations into an endogenous PPO2 gen (StPPO2) via CRISPR/Cas9, where the target region includes the copper binding site (CuA) (p6), resulting in plants with reduced PPO activity. Accordingly, claim15 is anticipated by González.
Claim 18 depends from claim 15 and recites that the mutation is introduced by genome editing.
González discloses introducing the mutation via CRISPR/Cas9 genome editing. Accordingly, claim 18 is anticipated by González.
Claim 19 depends from claim 15 and recites introducing a Cas nuclease and a guide RNA targeting the PPO1 or PPO2 gene.
González discloses introducing Cas9 and sgRNAs targeting StPPO2 to generate the mutations. Accordingly, claim 19 is anticipated by González.
Claims 1-3, 5, 7-10, and 14 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Beecher ( Brian S. Beecher et. al., Genetic mapping of new seed-expressed polyphenol oxidase genes in wheat (Triticum aestivum L.). Theor Appl Genet (2012) 124:1463–1473).
Claim 1 recites a modified plant, or a progeny thereof, having reduced polyphenol oxidase (PPO) activity, the plant comprising a mutation in at least one endogenous PPO1 or PPO2 gene, wherein the mutation is in a region of the endogenous PPO1 or PPO2 gene that encodes a copper binding domain of a PPO polypeptide.
The specification defines “modified” as containing changes or variations from a “natural or native state”. Under BRI, the “natural or native state” is not a single fixed sequence for a crop species, because multiple naturally occurring alleles of a gene exist within the species gene pool, and different cultivars/lines carry different alleles. In crop plants, domestication and routine cultivation/breeding/selection change allele frequencies and fix particular allelic variants in different varieties. Accordingly, when a reference/baseline allele is selected, an alternative allelic variant disclosed in the prior art constitutes a “variation from the natural or native state” relative to that baseline and therefore falls within the specification’s definition of “modified”, even if the variant arose through conventional breeding/selection rather than transgenesis.
Beecher discloses a wheat PPO gene system in which allelic variation in a kernel-expressed PPO locus includes mutations located within or directly associated with a catalytic copper-binding region (CuA) and associates such alleles with reduced kernel PPO activity relative to alternative alleles (p1467, fig 2).
Therefore, Beeches discloses (1) a wheat plant (and progeny) comprising an endogenous PPO gene allele (PPL-D1a), (2) having a mutation within the region encoding a catalytic copper-binding site (CuA) (e. g., L202V), and (3) associated with reduced PPO activity relative to plants lacking that mutation/allele (e.g., the lines describing PPO-D1a as low-activity vs PPO-1b and/or the QTL peak at PPO-D1). Accordingly, Beecher anticipates claim 1.
Claim 3 depends from claim 1 and recites that “the mutation is an insertion, a deletion, or a substitution of one or more nucleotides”.
Beecher discloses a wheat plant/cultivar comprising an endogenous PPO allele PPO-D1a that differs from another allele from SNPs, which constitute a substitution within the region encoding a catalytic copper-binding site (CuA) (e., g., L202V), as recited in claim 3. Accordingly, Beecher discloses the mutation type required by claim 3, and claim 3 is anticipated.
Claim 5 depends from claim 1, and recites the PPO activity in the grain of the plant is reduced relative to the PPO activity in grain without the mutation.
Beecher discloses that wheat lines carrying the PPO-D1a (low-PPO) exhibit reduced PPO activity relative to lines carrying PPO-D1b, the high-PPO alleles (p1471), which meets the limitation that “PPO activity in the grain…is reduced relative to grain without the mutation.”
Claim 7 depends from claim 1, and recites wherein browning of processed grain of the modified plant is reduced compared to processed grain of a control plant not comprising the mutation.
Beecher discloses that polyphenol oxidase (PPO) enzymatic activity is a major cause in time-dependent discoloration (browning) in wheat dough products (abstract and introduction). PPO causes the hydroxylation and dehydrogenation of these phenolic compounds to form reactive ortho-quinone products. These polyphenol oxidase then either self-polymerize or react with accessible nucleophilic moieties to form large light absorbing melanin which cause darkening and discoloration (p1463-1464). A wheat plant having reduced PPO activity (as required by claim 1) would yield processed grain products with reduced browning/discoloration relative to a control plant lacking the PPO-reducing condition. Accordingly, claim 7 is anticipated by Beecher.
Claims 8-10 depend from claim 1. As set forth in the 102 rejection of claim 1, Beecher discloses that the plant is wheat (Triticum aestivum L.). wheat is a member of the Triticeae tribe. Therefore, Beecher meets the additional limitation of claim 8 “where in the plant is in the Triticeae tribe”. Because Beecher discloses the plant is wheat, Beecher also meets the additional limitations of claims 9-10, which recite that the plant is “a wheat, barley, or rye plant” (claim 9) and specifically “a wheat plant” (claim 10). Accordingly, Beecher anticipates claims 8-10.
Claim 14 depends from claim 1 and recites a plant part, plant cell, or seed of the modified plant of claim 1.
Beecher further discloses analysis of Kernel/seed in connection with PPO activity (e.g., “Kernel PPO analysis” performed on whole kernels/seeds; and PPO gene described as “see-expressed” and “expressed in developing wheat kernels” mentioned in abstract and introduction), and thus discloses seed of the plant meeting claim 1. Therefore, Beecher anticipates the subject matter of claim 14 at lease as to the recited “seed” embodiment. Accordingly, claim 14 is anticipated by Beecher.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1, 2, 15 and 16 are rejected under 35 U.S.C. §103 as being unpatentable over Liu (Congcong Liu et. al., In-depth genetic analysis reveals conditioning of polyphenol oxidase activity in wheat grains by cis regulation of TaPPO2A-1 expression level, Genomics 112 (2020) 4690–4700) in view of Beecher (Theor Appl Genet (2012) 124:1463–1473) and Zhang (Shujuan Zhang et. al., CRISPR/Cas9-mediated genome editing for wheat grain quality improvement, Plant Biotechnology Journal (2021) 19, pp. 1684–1686).
Claim 1 recites a modified plant, or a progeny thereof, having reduced polyphenol oxidase (PPO) activity, the plant comprising a mutation in at least one endogenous PPO1 or PPO2 gene, wherein the mutation is in a region of the endogenous PPO1 or PPO2 gene that encodes a copper binding domain of a PPO polypeptide.
Liu teaches TaPPO2A-1 expression is the key factor in regulation of PPO activity in
wheat grains (abstract and introduction), and promoter haplotypes/SNPs can change expression and PPO activity.
Liu does not teaches that the mutation in endogenous PPO1/ PPO2 gene that encodes a copper binding domain of a PPO polypeptide.
Beecher teaches a wheat PPO gene system in which allelic variation in a kernel-expressed PPO locus includes mutations located within or directly associated with a catalytic copper-binding region (CuA) and associates such alleles with reduced kernel PPO activity relative to alternative alleles (p1467, fig 2).
Zhang teaches using genome editing in wheat to introduce targeted mutations in grain-quality genes, including multiple PPO genes, to obtain plants with reduced PPO function /activity (p1684-1685).
It would have been obvious to a POSITA, motivated by Liu teaches TaPPO2A-1 expression is the key factor in regulation of PPO activity in wheat grains, and Beecher’s teaching that the CuA copper-binding region is catalytic and highly conserved, to apply Zhang’s indel editing approach to generate loss-of-function-type mutations in the copper-binding -domain-encoding region of wheat TaPPO2A-1 gene to reduce PPO activity. Liu provides the target rationales and Zhang provides the method that correspond to claim 1. Accordingly, claim 1 is obvious over Liu, in view of Beecher and Zhang.
Claim 2 further limits the plant of claim 1 by reciting that “the endogenous PPO1 and PPO2 gene comprises a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 22, 23, 28, or 29”
Liu teaches TaPPO2A-1 (TraesCS2A01G468200.1, Table S10. Protein family analysis of the 20 PPO genes using Pfam 32.0.) is exactly the same locus as SEQ ID NO:1 (TraesCS2A01G468200, paragraph 0036). (Chromosomal locations of the two locus are list below).
Accordingly, claim 2 is obvious over Liu, in view of Beecher and Zhang.
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Claim 15 recites a method of reducing PPO activity in a plant, the method comprising: introducing a mutation in at least one endogenous PPO1 or PPO2 gene, wherein the mutation is in a region of the endogenous PPO1 or PPO2 gene that encodes a copper binding domain of a PPO polypeptide.
Liu teaches TaPPO2A-1 expression is the key factor in regulation of PPO activity in
wheat grains (abstract and introduction), and promoter haplotypes/SNPs can change expression and PPO activity. Liu does not teaches that the mutation in endogenous PPO1/ PPO2 gene that encodes a copper binding domain of a PPO polypeptide.
Beecher teaches a wheat PPO gene system in which allelic variation in a kernel-expressed PPO locus includes mutations located within or directly associated with a catalytic copper-binding region (CuA) and associates such alleles with reduced kernel PPO activity relative to alternative alleles (p1467, fig 2).
Zhang teaches using genome editing in wheat to introduce targeted mutations in grain-quality genes, including multiple PPO genes, to obtain plants with reduced PPO function /activity (p1684-1685).
It would have been obvious to a POSITA, motivated by Liu teaches TaPPO2A-1 expression is the key factor in regulation of PPO activity in wheat grains, and Beecher’s teaching that the CuA copper-binding region is catalytic and highly conserved, to apply Zhang’s indel editing approach to generate loss-of-function-type mutations in the copper-binding -domain-encoding region of wheat TaPPO2A-1 gene to reduce PPO activity. Liu provides the target rationales and Zhang provides the method that correspond to claim 15. Accordingly, claim 15 is obvious over Liu, in view of Beecher and Zhang.
Claim 16 depends from claim 15 recites the endogenous PPO1 or PPO2 gene comprises a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 22, 23, 28, or 29.
Because Liu teaches wheat TaPPO2A-1 gene sequence information and identifies the TaPPO2A-1 gene with 100% identity of SEQ ID NO:1 (check sequence information above). Claim 16 is obvious to Liu, in view of Beecher and Zhang.
Claims 4, 6, 11-13, and 15, 17-33 are rejected under 35 U.S.C. §103 as being unpatentable over Beecher (Theor Appl Genet (2012) 124:1463–1473) in view of Zhang (Plant Biotechnology Journal (2021) 19, pp. 1684–1686).
Claim 1 as the teachings of Beecher is discussed above.
Claim 4 depends from claim 1 and recites the mutation introduces a frameshift mutation, a pre-mature stop codon, or an in-frame deletion in the PPO1 or PPO2 gene.
Beecher does not teaches the mutation introduces a frameshift, pre-mature stop codon, or an in-frame deletion in PPO.
Zhang teaches genome editing in wheat targeting grain-quality genes including PPO, and show indel outcomes at edited targets such as +1 insertions and -1 bp, -2bp, -3bp deletions for PPO edits (fig 1 (b)). +1bp/-1 bp/-2 bp insertions are classic frameshift-inducing edits, and often leading to premature stop codon.
It would have been obvious to a POSITA, motivated by Beecher’s teaching that PPO contributes to dough discoloration and that the CuA copper-binding region is catalytic and highly conserved, to apply Zhang’s indel editing approach to generate loss-of-function-type mutations in the copper-binding -domain-encoding region of a wheat PPO gene to reduce PPO activity. Beecher provides the target rationales and Zhang provides the method that correspond to claim 4. Accordingly, claim 4 is obvious over Beecher in view of Zhang.
Claim 5 as the teachings of Beecher is discussed above.
Claim 6 depends from claim 5 and recites the modified plant of claim 5, wherein the PPO activity is reduced by at least 70%, at least 80%, or at least 90% relative to the PPO activity in grain without the mutation.
Beecher teaches kernel PPO activity is a major cause of time-dependent discoloration of wheat dough products (p1463-1464); multiple kernel-expressed PPO loci/alleles contribute to grain PPO activity (p1464).
Beecher does not teaches PPO activity is reduced by 70% to 90% relative to the PPO activity in grain without the mutation.
Zhang teaches using genome editing in wheat to introduce targeted mutations in grain-quality genes, including multiple PPO genes, to obtain plants with reduced PPO function /activity.
A POSITA motivated by Beecher to reduce kernel PPO activity to improve discoloration would have found it obvious to apply Zhang’s editing approach to PPO genes, and routinely optimize editing/copy number/allele selection to achieve strong reductions, including ≥70%-90%, as a matter of result-effective optimization. Claim 6 is obvious in view of Beecher and Zhang.
Claim 10 as the teachings of Beecher are discussed above.
Claim 11 depends from claim 10 recites the plant comprises a mutation in at least two, three, four, five, or six endogenous PPO1 or PPO2 genes selected from PPO2A-1, PPO2A-2, PPO2B-1, PPO2B-2, PPO2B-3, PPO2D-1, and PPO2D-2.
Beecher does not teach modifying at least two, three, four, five, or six endogenous PPO1 or PPO2 genes selected from PPO2A-1, PPO2A-2, PPO2B-1, PPO2B-2, PPO2B-3, PPO2D-1, and PPO2D-2.
Zhang teaches using genome editing to introduce targeted loss-of-function mutations in wheat grain-quality genes, including PPOs.
It would have been obvious to use Zhang’s CRISPR/Cas9 method to make the PPO-reducing PPO genes modification taught by Beecher because it is a known, which genes to target, and how many genes to modify, is routine technique and would predictably produce plants with reduced PPO activity.
Accordingly, claim 11 is obvious over Beecher in view of Zhang.
Claim 12 depends from claim 11 recites the plant comprises a mutation in PPO2A-1, PPO2A-2, PPO2B-1, PPO2B-2, PPO2B-3, PPO2D-1, and PPO2D-2.
Beecher does not teaches that the plant comprises mutations in each of the listed PPO genes (PPO2A-1, PPO2A-2, PPO2B-1, PPO2B-2, PPO2B-3, PPO2D-1, and PPO2D-2). Given the combined teachings of Beecher and Zhang, it would have been obvious to mutate all of the listed PPO genes as a routine optimization to further reduce PPO activity. Accordingly, claim 12 is obvious over Beecher in view of Zhang.
Claim 13 depends from claim 10 recites the wheat plant is of the variety Fielder, Guardian, or Steamboat.
Neither Beecher nor Zhang teaches a variety of Fielder, Guardian, or Steamboat. Fielder, Guardian, and Steamboat varieties and information can be found at the Agricultural Experiment Station of Colorado State University (https://webdoc.agsci.colostate.edu/csucrops/reports/winterwheat/wheatreport_2022.pdf, accessed 12/29/2025); Fielder variety and information can be found from Agricultural Extension Service University of Idaho (https://washingtoncrop.com/documents/Wheat/Spring/Soft%20White/Fielder.pdf, accessed 12/29/2025). It would have been obvious to implement the modified PPO genotype taught by Beecher and Zhang in any known wheat variety, including Fielder, Guardian, or Steamboat, because selecting among known wheat varieties for introducing a known genetic modification is a predictable substitution of one known cultivar for another. And the teaching of reducing PPO activity would reasonably be expected to apply across wheat genetic backgrounds. Accordingly, claim 13 is obvious by Beecher and Zhang.
Claim 15 recites a method of reducing PPO activity in a plant, the method comprising: introducing a mutation in at least one endogenous PPO1 or PPO2 gene, wherein the mutation is in a region of the endogenous PPO1 or PPO2 gene that encodes a copper binding domain of a PPO polypeptide.
Beecher teaches wheat PPO genes and PPO polypeptides, including that PPO activity depends on conserved copper binding catalytic domains (p1463-1464). Beecher does not teach the method to mutate at least one endogenous PPO gene.
Zhang teaches reducing PPO activity in wheat by introducing mutations into endogenous PPO genes using CRISPR/Cas genome editing, and evaluating reduced grain/kernel PPO activity in the resulting mutants (p1684-1685).
It would have been obvious to one of ordinary skill to target the CRISPR/Cas edits taught by Zhang to a region encoding the copper-binding domain as a predictable way to reduce PPO enzymatic activity, since disrupting a function-critical catalytic domain would be expected to diminish PPO activity. Claim 15 is obvious over Beecher and Zhang.
Claim 17 depends from claim 15 recites the PPO activity in the grain of the plant is reduced relative to the PPO activity in grain without the mutation.
Because Zhang teaches that introducing PPO mutations leads to reduced PPO activity in grain/kernels (p1685, fig 1). Claim 17 is obvious over Beecher and Zhang.
Claim 18 depends from claim 15 recites the mutation is introduced by genome editing.
Beecher does not teaches genome editing.
Because Zhang expressly teaches introducing the mutation by genome editing. Claim 18 is obvious in view of Beecher and Zhang.
Claim 19 depends from claim 15 recites the method comprises introducing a Cas nuclease and a guide RNA targeting the PPO1 or PPO2 gene.
Beecher does not teaches Cas nuclease system.
Because Zhang teaches a CRISPR/Cas approach that uses a Cas nuclease and a guide RNA targeting the PPO gene (fig 1). Claim 19 is obvious in view of Beecher and Zhang.
Claim 20 depends from claim 15 recites the guide RNA comprises the nucleotide sequence of SEQ ID NO: 34.
Beecher does not teaches genome editing system.
Because once Beecher provides the PPO target sequences and Zhang teaches CRISPR/Cas editing of PPO in wheat, selecting a particular guide RNA sequence (e. g., SEQ ID NO:34) that targets PPO1/PPO2 would have been a routine design choice/optimization within ordinary skill to achieve the taught PPO-reducing edits. Claim 20 is obvious over Beecher and Zhang.
Claim 21 depends from claim 15 recites the plant is in the Triticeae tribe.
Zhang teaches introducing genome-edited PPO mutations in wheat to reduce PPO activity, and wheat is a member of the Triticeae tribe, therefore limiting the method to a Triticeae plant is an obvious limitation in view of Zhang’s wheat example.
Claim 22 depends from claim 15 recites the plant is a wheat, barley, or rye plant.
Beecher teaches PPO genes and PPO-related quality traits in cereal plants, and Zhang teaches the claimed PPO-reduction method in wheat. It would have been obvious to apply the same genome-editing approach to other closely related Triticeae crops such as barley or rye to reduce PPO activity, because PPO genes are conserved in these cereals and the results would be expected.
Claim 23 depends from claim 15 recites the plant is a wheat plant. For the same reasons as claim 15, because Zhang expressly teaches performing the PPO-reduction genome-editing method in wheat. Claim 23 is obvious over Beecher and Zhang.
Claim 24 depends from claim 23 recites the mutation is introduced in at least two, three, four, five, or six endogenous PPO1 or PPO2 genes selected from PPO2A-1, PPO2A-2, PPO2B-1, PPO2B-2, PPO2B-3, PPO2D-1, and PPO2D-2.
Beecher teaches that wheat has multiple PPO genes/copies contributing to PPO activity in polyploid wheat. Beecher does not teaches the listed six endogenous genes. Zhang teaches using genome editing (CRISPR/Cas) to introduce mutations into endogenous PPO genes in wheat to reduce PPO activity. It would have been obvious to introduce mutations into two or more of the listed endogenous PPO1/PPO2 genes as a routine optimization to further reduce overall PPO activity by addressing gene redundancy.
Claim 25 depends from claim 23 recites the mutation is introduced in PPO2A-1, PPO2A-2, PPO2B-1, PPO2B-2, PPO2B-3, PPO2D-1, and PPO2D-2.
For the same reason as claim 24, mutating all of the listed PPO1/PPO2 genes (PPO2A-1, PPO2A-2, PPO2B-1, PPO2B-2, PPO2B-3, PPO2D-1, and PPO2D-2) would have been an obvious extension of mutating multiple PPO genes in a polyploid crop to maximize reduction of PPO activity. Claim 25 is obvious over Beecher and Zhang.
Claim 26 depends from claim 23 recites the wheat plant is of the variety Fielder, Steamboat, or Guardian.
Neither Beecher nor Zhang teaches a variety of Fielder, Guardian, or Steamboat. Fielder, Guardian, and Steamboat varieties and information can be found at the Agricultural Experiment Station of Colorado State University (https://webdoc.agsci.colostate.edu/csucrops/reports/winterwheat/wheatreport_2022.pdf, accessed 12/29/2025); Fielder variety and information can be found from Agricultural Extension Service University of Idaho (https://washingtoncrop.com/documents/Wheat/Spring/Soft%20White/Fielder.pdf, accessed 12/29/2025). It would have been obvious to implement the modified PPO genotype taught by Beecher and Zhang in any known wheat variety, including Fielder, Guardian, or Steamboat, because selecting among known wheat varieties for introducing a known genetic modification is a predictable substitution of one known cultivar for another. And the teaching of reducing PPO activity would reasonably be expected to apply across wheat genetic backgrounds. Accordingly, claim 26 is obvious by Beecher and Zhang.
Claim 27 depends from claim 1 recites a method of producing a plant having reduced PPO activity, the method comprising: (a) crossing the plant of claim 1 with itself or another plant to produce seed; (b) growing one or more progeny plants from the seed; and (c) selecting a progeny plant comprising the mutation to produce a plant having reduced PPO activity.
Beecher teaches wheat plants with reduced PPO activity and recognizes PPO traits are inherited (p1463, abstract and introduction). Beecher does not teach the method of producing a plant having reduced PPO activity. Zhang teaches generating wheat plants with PPO gene mutation and evaluating plants with reduce PPO activity. It would have been obvious to one of ordinary skill to cross such a reduced-PPO plant (mutant plant) with itself or another wheat plant, grown progeny, and select progeny carrying the PPO mutation, because these are routine breeding steps used to propagate and fix a desired, heritable mutation/trait and to obtain plants having reduced PPO activity. Claim 27 is obvious over Beecher and Zhang.
Claim 28 depends from claim 27 recites the method comprising: (c) crossing the progeny plant with itself or another plant; and (d) repeating steps (b) and (c) for an additional 0-7 generations to produce a plant having grain with reduced PPO activity.
Neither Beecher nor Zhang teaches or suggests any breeding practice (e.g., crossing,
Backcrossing, or selection) to produce the claimed plant.
Further in view of ordinary breeding practice, repeating the grow/selection and optional crossing steps for additional generations (0-7) to obtain grain with reduced PPO activity is an obvious, routine way to stabilize and introgress the PPO mutation and to recover lines with the desired grain phenotype. Claim 28 is obvious in view of Beecher and Zhang.
Claim 29 depends from claim 1 recites a crop comprising a plurality of the plants of claim 1 planted together in an agricultural field.
It would have been obvious to plant a plurality of the obvious modified plats together in an agricultural field to form a crop, because this is the routine and intended manner of cultivating wheat plants for agricultural production. Claim 29 is obvious.
Claim 30 depends from claim 1 recites a commodity plant product prepared from the plant of claim 1, or a part thereof, wherein the product has reduced browning.
Beecher teaches that PPO activity is responsible for enzymatic browning; Beecher does not teach the method to modify PPO; Zhang teaches plant with PPO mutations and reduced PPO activity; therefore, it would have been obvious that a commodity product prepared from such plants would exhibit reduced browning. Claim 30 is obvious in view of Beecher and Zhang.
Claim 31 depends from claim 30 recites the product is grain, flour, a baked good, cereal, pasta, a beverage, livestock feed, biofuel, straw, a construction material, or starch.
The limitation of claim 31 is the listed items (grain, flour, a baked good, cereal, pasta, a beverage, livestock feed, biofuel, straw, a construction material, or starch) are conventional commodity products routinely prepared from wheat, and specifying them does not add a patentable distinction. Claim 31 is obvious.
Claim 32 depends from claim 1 recites a method for producing a commodity plant product, the method comprising processing the plant of claim 1, or a part thereof, to obtain the product.
Beecher teaches PPO is responsible for enzymatic browning, Zhang teaches producing wheat plants with PPO mutations and reduced PPO activity. It would have been obvious to process the obvious modified plant (or plant part) to obtain a commodity product, because processing harvested wheat into products is routine and the resulting products would be expected to show reduced browning due to reduced PPO. Claim 32 is obvious.
Claim 33 depends from claim 32 recites the commodity plant product is grain, flour, a baked good, cereal, pasta, a beverage, livestock feed, biofuel, straw, a construction material, or starch. The recited products (grain, flour, a baked good, cereal, pasta, a beverage, livestock feed, biofuel, straw, a construction material, or starch) are standard outputs of wheat processing, and specifying them does not add a patentable distinction. Claim 33 is obvious.
Claims 34-37 are rejected under 35 U.S.C. §103 as being unpatentable over Zhang (Plant Biotechnology Journal (2021) 19, pp. 1684–1686).
Claim 34 recites a guide RNA for editing a PPO1 or PPO2 gene comprising the nucleotide sequence of SEQ ID NO: 34.
Zhang teaches that one sgRNA is designed to target conserved sites in the third exon of all three PPO homeologs, i.e., using a guide RNA to edit PPO (p1686). Therefore, providing a guide RNA comprising a particular target sequence (SEQ ID NO:34) for editing PPO1/PPO2 would have been an obvious matter of routine guide design to carry out the PPO-editing taught by Zhang. Claim 34 is obvious over Zhang.
Claim 35 depends from claim 34 recites an expression cassette or vector encoding the guide RNA of claim 34.
Zhang’s CRISPR editing approach necessarily uses an expression cassette/vector to express the sgRNA and the delivery system (p1684). Encoding the guide RNA of claim 34 in a cassette or vector is an obvious implementation detail. Claim 34 is obvious over Zhang.
Claim 36 depends from claim 34 recites a plant cell comprising a Cas9 nuclease and the guide RNA of claim 34.
Zhang’s method of CRISPR editing requires a plant cell that contains (at least transiently) a Cas9 nuclease and the sgRNA targeting PPO. Thus, a plant cell comprising Cas9 and the guide RNA is obvious/inherent in practicing Zhang. Claim 36 is obvious.
Claim 37 depends from 36 recites the plant cell of claim 36, wherein the plant cell is wheat plant cell, a rye plant cell, or a barley plant cell.
Zhang’s disclosure is in wheat, making a wheat plant cell embodiment obvious. Extending the same Cas9/sgRNA PPO-editing system to closely related cereal plant cells such as rye or barley would have been an obvious variation with an expected results editing the PPO gene.
Conclusion
No claims are allowed.
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/YANXIN SHEN/Examiner, Art Unit 1663
/WEIHUA FAN/Primary Examiner, Art Unit 1663