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 1-5 are pending.
Claims 1-5 are examined on the merits.
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 are rejected under 35 U.S.C. §103 as being unpatentable over Silva Junior (Carlos Alberto Silva Junior et. al., Annals of Applied Biology (2021)178, pp135–148) in view of Xie (L. Xie et. al., Plant Growth Regulation (2013) 69:295–303), further in view of Bassett (Carole L. Bassett et. al., Journal of America Society for Horticultural Science (2014)139(4):487–494), An (Jian-Ping An et. al., Journal of Experimental Botany (2020) Vol. 71, No. 10 pp. 3094–3109), and Huo (Liuqing Huo et. al., Frontiers in Plant Science (2020) Volume 11, article 423 pp1-14).
Claim 1 is drawn to a method for promoting apple anthocyanin biosynthesis, comprising infecting an apple tissue with a recombinant Agrobacterium carrying an overexpression vector containing an MdPHY7 gene having the nucleotide sequence of SEQ ID NO: 2, which is MdPHY7 gene.
Silva Junior teaches that Arabidopsis “phyB1 and phyB2 mutants exhibited lower anthocyanin content” (page 141 right column first full paragraph), indicating that loss of phyB function reduce anthocyanin accumulation. Thus, Silva Junior demonstrates phyB positively regulates anthocyanin synthesis.
Silva Junior further teaches that the phyB family is a target molecule for plant breeding and that manipulation of phyB (including overexpression) results in agronomically important traits, including improved tolerance to environmental stress (page 135 Abstract), providing motivation for a POSITA to genetically manipulate phyB-family genes.
Silva Junior does not teach which specific phyB-family gene should be manipulated.
Xie teaches that increasing anthocyanin accumulation in apple peel/skin improves coloration and that treatments such as 5-aminolevulinic acid (ALA) increase anthocyanin accumulation in apple fruit tissues (page 295 abstract). Thus, Xie establishes the recognized goal in the art of promoting anthocyanin biosynthesis in apple tissue to improve apple coloration.
Xie does not teach 5-aminolevulinic acid (ALA) increases anthocyanin accumulation through MdPHY7, while Silva Junior provides evidence that phyB function is linked to anthocyanin accumulation, which would have suggested to a POSITA that increasing phyB activity/signaling would be a reasonable strategy to promote anthocyanin accumulation.
Bassett teaches an apple phytochrome gene, MdPHYB (MDP0000285110) (supplementary table 1) and provides sequence information and primers (MdPhyBF1: 5’-TCTGGTTCACGATCTCATTT-3’; MdPhyBR1: 5’-TTGGAGGGCTAGACACAGACA-3’) (supplementary table 2)enabling a person of ordinary skill in the art to isolate the gene. The sequence obtained using the primers disclosed in Bassett corresponds to a nuclei acid that includes the sequence of SEQ ID NO:2 (100% identity over the overlapping region). Therefore, Bassett teaches a nucleic acid comprising a sequence overlapping and identical to SEQ ID NO:2 (see alignment).
An teaches transient gene expression in apple tissues using Agrobacterium delivery of plant expression constructs to evaluate gene regulatory effects under light conditions (page 3095-3096, Material and method).
Huo teaches stable Agrobacterium -mediated transformation of apple callus tissue using a CaMV35S-driven overexpression construct (e.g., MdATG9 in pCambia2300), and selection of transformed apple callus cells (page 2-3, Materials and methods).
A person of ordinary skill in the art would have been motivated to overexpress an apple phytochrome gene in apple tissue using Agrobacterium in order to modulate anthocyanin, with a reasonable expectation of success.
Claim 2 is drawn to the method of claim 1, wherein a site for promoting apple anthocyanin biosynthesis is selected from the group consisting of a peel, a leaf, and a callus of an apple.
An teaches Agrobacterium-mediated transient expression in apple calli, leaves, and fruit (including peel) (page 3096 Genetic transformation). Therefore, applying the Agrobacterium overexpression method to peel, leaf, or callus is a predictable use of known techniques across known apple tissue.
Because Xie recognizes increasing anthocyanin in apple as a desirable goal for better apple quality; Silva Junior discloses phyB activity is associated with increased anthocyanin levels; Bassett discloses apple phytochrome gene; An and Huo provide routine methods of Agrobacterium-mediated introduction and overexpression of genes in apple tissues and callus.
Taken together, a POSITA seeking to achieve Xie’s recognized objective of increasing anthocyanin in apple tissues would have been motivated by silva Junior’s teaching that phyB function is positively associated with anthocyanin accumulation (as evidenced by reduced anthocyanin in phyB mutants) to increase phytochrome-family activity in apple, and Bassett provides an apple phytochrome nucleic acid comprising a region identical to SEQ ID NO: 2 for implementing that approach. According, the prior arts support conclusion that manipulating an apple phytochrome gene corresponding to SEQ ID NO:2 would have been a motivated and reasonable avenue toward promoting anthocyanin biosynthesis in apple tissue.
A person of ordinary skill in the art would have been motivated to obtain an apple phytochrome gene (including the fragment corresponding to SEQ ID NO: 2) using the MdPHyB gene taught by Bassett and insert that gene into an overexpression vector delivered into apple tissue via Agrobacterium to modulate anthocyanin biosynthesis, with a reasonable expectation of success.
The claimed invention in claims 1 and 2 as a whole is prima facie obvious over the combined teachings of the prior arts above.
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Claims 3-5 are rejected under 35 U.S.C. §103 as being unpatentable over Wang (Shuai Wang et. al., THE PLANT CELL (2022) 34: pp1980–2000), in view of Li (Yajun Li et. al., Plant Physiology and Biochemistry (2020) 147, pp272-279), and Silva Junior (2021), Bassett (2014), and An (2020).
Claim 3 is drawn to an interfering recombinant plasmid, wherein the interfering recombinant plasmid is inserted with an apple anthocyanin biosynthesis-related MdPHY7 gene fragment having the nucleotide sequence set forth in SEQ ID NO: 3.
For purposes of applying prior art, an “interfering recombinant plasmid” is interpret as a recombinant plasmid comprising an inserted MdPHY7 nucleic-acid fragment (SEQ ID NO:3) configured to reduce/inhibit MdHY7 expression in apple tissue.
Wang teaches preparing a TRV-VIGS construct comprising an inserted fragment corresponding to the target apple gene (i. e., an interfering/silencing construct) and using it for gene knockdown in apple tissue (page 1991 first paragraph, page 1996 last paragraph).
Li teaches that selecting an inserted ~300-500 bp target gene fragment is standard /expected for VIGS (page 273, Construction of VIGS vector), making the claimed 500-nt fragment (SEQ ID NO:3) an obvious selection from the known MdPHY7 sequence (SEQ ID NO: 2) for use in the interfering plasmid.
Silva Junior provides the rational to choose a phytochrome/light-signaling gene as a target affecting anthocyanin (page 141 right column paragraph 2), making MdPHY7 (teaches by Bassett, supplementary table 2) an obvious candidate target for silencing. Accordingly, a POSITA would have found it obvious to make an interfering plasmid comprising a fragment of MdPHY7 (including a routine~500 nt fragment, such as SEQ ID NO:3) to suppress MdPHY7 expression.
Claim 4 is drawn to a method for inhibiting apple anthocyanin biosynthesis, comprising the following steps: infecting an apple tissue with a recombinant Agrobacterium strain carrying the interfering recombinant plasmid according to claim 3.
Wang teaches Agrobacterium-mediated infiltration of apple fruits with a TRV-VIGS (interfering) construct to achieve gene silencing/knockdown in apple tissue (page 1991 paragraph 1).
Claim 5 is drawn to the method according to claim 4, wherein a site for inhibiting apple anthocyanin biosynthesis is selected from the group consisting of a peel, a leaf, and a callus of an apple.
An teaches Agrobacterium-mediated transient expression in apple calli, leaves, and fruit (including peel) (page 3096 Genetic transformation), supporting that callus, apple peel and leaf are conventional apple tissue for Agrobacterium-based constructs.
A POSITA would have been motivated to use Wang’s established Agrobacterium-delivered TRV-VIGS platform in apple fruit/peel (including apple callus and leaf by An) to knock down a different apple gene, where Silva Junior teaches phyB gene is positively linked to anthocyanin synthesis in apple, providing a reasonable expectation that silencing MdPHY7 would inhibit anthocyanin biosynthesis. Selecting a ~500 nt target fragment is a routine VIGS design choice as taught by Li.
The claimed invention in claims 3-5 as a whole is prima facie obvious over the combined teachings of the prior arts above.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to YANXIN SHEN whose telephone number is (571)272-7538. The examiner can normally be reached Monday-Friday.
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/YANXIN SHEN/
Examiner, Art Unit 1663
/Amjad Abraham/ SPE, Art Unit 1663