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 .
Status of the Claims
Claims 2-6 and 16-17 are canceled.
Claims 1, 7-15, and 18 are pending.
The objections to claims 13 and 17 for minor informalities have been withdrawn in view of Applicant’s amendment to claim 13 and cancellation of the claim 17.
The rejections to claims 2 and 4 under 35 USC § 112(b) have been withdrawn in view of Applicant’s cancellation of the claims.
Claims 1, 7-12, and 14 are rejected.
Claims 13, 15, and 18 are objected to.
Claim Objections
Claims 13, 15, and 18 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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, 7-12, and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Osakabe (Osakabe, Y., Liang, Z., Ren, C., Nishitani, C., Osakabe, K., Wada, M., ... & Nagamangala Kanchiswamy, C. (2018). CRISPR–Cas9-mediated genome editing in apple and grapevine. Nature Protocols, 13(12), 2844-2863), Tricoli(b) (Tricoli, D. (2019). Grape protoplast isolation and regeneration of plants for use in gene editing technology. 2019 research progress report), Eriksson (Eriksson, Tage R. "Protoplast isolation and culture." Plant protoplasts. Crc Press, 2018. 1-20), and Bertini (Bertini, E., Tornielli, G. B., Pezzotti, M., & Zenoni, S. (2019). Regeneration of plants from embryogenic callus-derived protoplasts of Garganega and Sangiovese grapevine (Vitis vinifera L.) cultivars. Plant Cell, Tissue and Organ Culture (PCTOC), 138(2), 239-246).
This is a modified rejection necessitated by Applicant’s amendments.
Claim 1 is drawn to a method for the generation of genome edited protoplasts from clonally propagated plant tissue for generation of whole plants, wherein the method comprises the steps of:
a) providing a cell suspension from clonally propagated plant tissue selected from the group consisting of embryogenic callus, non-embryogenic callus, and/or leaf cells;
b) contacting the cell suspension with an enzyme composition for digestion of a plant cell wall, wherein the enzyme composition comprises 0.5 to 3 % (w/v) cellulase, 0.1 to 0.6% (w/v) hemicellulase, and 0.2 to 0.5% (w/v) macerozyme R-10;
c) isolating a protoplast from the cell suspension and washing of the isolated protoplast with a wash solution, wherein the wash solution is osmotically adjusted to correspond to the osmotic values of the isolated protoplast, and wherein the concentration of mannitol is at most 0.4 M;
d) genome editing of the isolated protoplast by delivery of biological material into the isolated protoplast; and
e) generating mini-callus colonies from the genome edited protoplast,
wherein the clonally propagated plant tissue is selected from the group consisting of grapevine and potato,
and wherein the method further comprises a step f) generating whole genome edited plants from the mini-callus colonies by culturing the mini-callus colonies to form into embryos and eventually to germinate into whole plants, wherein step f comprises culturing the mini-callus colonies in culture medium comprised of auxins, cytokinins, and optionally about 1 g/L activated charcoal, for at least 3 weeks.
Claim 7 is drawn to the method according to claim 1, wherein the ratio auxins:cytokinins in the culture medium is at least about 1:1.
Claim 8 is drawn to the method according to claim 1, wherein the culture medium comprises between 1.8 to 2.6 µM 6-Benzyladenine (6-BAP), and between 2.5 to 11.5 µM 1-Naphthaleneacetic acid (NAA), and optionally between 0.7 to 1.2 µM kinetin.
Claim 9 is drawn to the method according to claim 1, wherein genome editing is done by CRISPR-Cas technology.
Claim 10 is drawn to the method according to claim 1, wherein the whole plant is a genome edited grapevine plant selected from the group consisting of the cultivars Chardonnay, Crimson S., Thompson S., Merlot, Glera, Malbec, and Sugraone.
Claim 11 is drawn to the method according to claim 1, wherein the delivery of biological material into the protoplasts is achieved by means of liposome- or polyethylene glycol- (PEG) infiltration, electroporation, or lipofection.
Claim 12 is drawn to the method according to claim 1, wherein the biological material is comprised of one or more selected from the group consisting of CRISPR-Cas components, ribonucleic protein (RNP), single guide RNA (sgRNA), a vector encoding a Cas nuclease, and the sgRNA.
Claim 14 is drawn to the method according to claim 1, wherein after the genome editing of the isolated protoplast by delivery of biological material into the protoplast, the protoplasts are kept at 24-26 °C for 16 to 60 hours before proceeding to step e).
Regarding claim 1, Osakabe discloses isolating grapevine protoplasts from embryogenic callus that is derived from ‘Chardonnay’ anthers and sub-cultured over weeks to obtain the induced callus (i.e. clonally propagated tissue that is embryogenic callus) (p. 2855, step i, and p. 2857-2858, step vii). Osakabe discloses transferring induced callus pieces into 150 mL flask filled with 50 mL of liquid induction medium, to produce small, newly generated embryogenic cell masses (i.e. the clonally propagated callus is provided in a cell suspension) (p. 2855, step i). Then, Osakabe teaches immersing grapevine embryogenic calli in cell wall digestion enzyme solution comprising 0.15% Macroenzyme R-10 and 1% cellulase (p. 2857, step viii, p. 2853) (i.e. contacting the cell suspension with an enzyme composition for digestion of a plant cell wall). Next, Osakabe teaches after digestion, filtering the protoplast and washing them with a wash solution (p. 2859, step xi) (i.e. isolating the protoplasts and washing them with a wash solution). Osakabe further teaches transforming the protoplasts via CRISPR-Cas9 RNP delivery (p. 2859, step xvi-xvii) (i.e. genome editing of the isolated protoplast by delivery of biological material into the isolated protoplast), and also teaches regenerating the protoplasts to form microcalli (p. 2859, step xxii-xxvi) (i.e. generating mini callus colonies from the genome edited protoplast).
Regarding claim 9, Osakabe discloses transforming the protoplasts via CRISPR-Cas9 RNP delivery (p. 2859, step xvi-xvii) (i.e. wherein genome editing is done by CRISPR-Cas technology).
Regarding claim 10, Osakabe discloses the genome edited grapevine plant is of the cultivar Chardonnay (p. 2848, Materials: Genome editing by direct delivery of CRISPR—Cas9 RNPs in apple and grapevine protoplasts).
Regarding claim 11, Osakabe discloses transforming the protoplast with the RNPs by adding PEG (p. 2859, step xvii) (i.e. the delivery of the biological material into the protoplasts is achieved by means of PEG).
Regarding claim 12, Osakabe discloses the biological material is RNP (p. 2859, step xvi-xvii).
Regarding claim 14, Osakabe discloses, after RNP delivery and before protoplast regeneration to form microcalli, an incubation step that occurs overnight at room temperature (RT) (p. 2859, step xix) (i.e. reasonably interpreted as wherein after the genome editing of the isolated protoplast by delivery of biological material into the protoplast, the protoplasts are kept at 24-26°C for 16 to 60 hours before proceeding to step e) generating mini callus colonies from the genome edited protoplast).
However, Osakabe does not explicitly teach:
Wherein the enzyme composition comprises 0.1 to 0.6% (w/v) hemicellulase, and 0.2 to 0.5% (w/v) macerozyme R-10 (remaining limitation of claim 1)
wherein the wash solution is osmotically adjusted to correspond to the osmotic values of the isolated protoplast, and wherein the concentration of mannitol is at most 0.4 M(remaining limitation of claim 1)
wherein the method further comprises a step f) generating whole genome edited plants from the mini-callus colonies by culturing the mini-callus colonies to form into embryos and eventually to germinate into whole plants, wherein step f comprises culturing the mini-callus colonies in culture medium comprised of auxins, cytokinins, and optionally about 1 g/L activated charcoal, for at least 3 weeks(remaining limitation of claim 1)
The method according to claim 1, wherein the ratio auxins:cytokinins in the culture medium is at least about 1:1 (claim 7)
The method according to claim 1, wherein the culture medium comprises between 1.8 to 2.6 pM 6-Benzyladenine (6-BAP), and between 2.5 to 11.5 pM 1-Naphthaleneacetic acid (NAA), and optionally between 0.7 to 1.2 pM kinetin (claim 8)
Regarding the remaining limitations of claim 1, In analogous art, Tricoli(b) teaches about grapevine protoplast isolation and specifically teaches the enzyme solution comprises 0.5% cellulase and 0.25% macroenzyme R-10 (p. 3). Tricoli(b) also teaches the protoplasts are washed in an osmotically adjusted wash solution comprising 0.4 M Mannitol (p.3). Furthermore, Tricoli(b) teaches culturing the grape suspension protoplast to stimulate the formation of mini calli (p. 3-9) and teaches about plans to test various media formulation to create embryogenic callus colonies and eventually regenerate embryos and plants from these protoplast-derived callus colonies (p. 9-10). Tricoli(b) teaches the some of the regeneration mediums to be tested include Picloram (Pic) and Thidiazuron (TDZ) (p. 9) (i.e. the culture medium is comprised of auxins and cytokinins). In other analogous art, Eriksson teaches the isolation of protoplasts has become routine as a result of the availability of a number of commercially available cell wall degrading enzymes, and the most extensively used include cellulases and hemicellulases, and pectinases including macroenzyme (p. 3-4, Table 1). Eriksson also teaches the source of tissue used in protoplast isolation not only determines what enzymes should be used but also the enzyme concentration and the treatment time, and it is necessary to adjust the enzyme concentration to optimize yields and viabilities of the isolated protoplasts (p. 5, ¶3). In other analogous art, Bertini teaches regenerating grapevine embryos and whole plants from protoplasts using a media comprising NAA and 6-BAP, and cultured for at least 3-4 months (p. 241, section titled Protoplast culture for somatic embryogenesis, and Fig. 3).
Regarding claims 7 and 8, Bertini teaches about the regeneration of grapevine plants from embryogenic callus-derived protoplasts (title). Specifically, Bertini describes observation of micro colonies about 40 days after protoplast isolation, teaching that embryogenesis was induced from callus derived protoplasts (p. 242, see cell division, microcolony formation, and somatic embryogenesis section). Bertini teaches the medium to induce somatic embryogenesis from the protoplast was supplemented with 2 mg/L NAA (i.e. an auxin) and 0.5 mg/L 6-BAP(i.e. a cytokinin), and were cultured for 3-4 months (i.e. micro colonies would have been present at 40 days and cultured in the media comprising 2 mg/L NAA and 0.5 mg/L 6-BAP) (p. 241, see protoplast culture for somatic embryogenesis section). Thus, regarding claim 7, Bertini teaches a ratio of 4:1 NAA (auxin) to 6-BAP (cytokinin), which encompasses the claimed ratio of auxin:cytokinin in the culture medium being at least about 1:1. Additionally, regarding claim 8, 2 mg/L NAA is equivalent to 10.7 uM and 0.5 mg/L equivalent to 2.2 uM, and therefore the medium taught be Bertini teaches the claimed medium that comprises between 1.8 to 2.6 uM 6-Benzyladenine (6-BAP), and between 2.5 to 11.5 uM 1- Naphthaleneacetic acid (NAA).
It would therefore have been obvious to a person of ordinary skill in the art to modify the invention of as taught by Osakabe to include the limitations of Tricoli(b), Eriksson, and Bertini to arrive at the instantly claimed method with a reasonable expectation of success because Tricoli(b) and Bertini are specifically directed to grapevine protoplast isolation and regeneration, and the teachings of Erikson are directed to general protoplast isolation, and one of ordinary skill in the art would could incorporate the limitations of Tricoli(b), Eriksson, and Bertini into the method taught by Osakabe without encountering any special technical obstacles. One having ordinary skill in the art would have been motivated to combine the teachings of Tricoli(b) with the teachings of Osakabe because Tricoli(b) teaches a method of grape protoplast isolation and regeneration of plants for use in gene editing technology (title), and teaches the importance of osmotically adjusting the medium to avoid implosion or explosion of the protoplasts (p. 3). Tricoli(b) specifically teaches successfully obtaining protoplast when washing the protoplasts in medium with 0.4 M Mannitol (p. 3). One having ordinary skill in the art would have been motivated to combine the teachings of Eriksson with the teachings of Osakabe because Eriksson teaches protoplast isolation has become routine, and all of the instantly claimed enzymes are commercially available and the most commonly employed in protoplast isolation (p. 3-4, Table 1). Furthermore, Eriksson teaches the source of tissue used in protoplast isolation not only determines what enzymes should be used but also the enzyme concentration and the treatment time, and it is necessary to adjust the enzyme concentration to optimize yields and viabilities of the isolated protoplasts (p. 5, ¶3). For this reason, it would be obvious to use the most commonly employed enzymes for protoplast isolation, including cellulase, hemicellulase, and macroenzyme, and adjust the concentrations to arrive at the instantly claimed method for the purpose of optimizing yields and viabilities of the isolated protoplasts. Additionally, one having ordinary skill in the art would have been motivated to combine the teachings of Bertini with the teachings of Osakabe because Bertini is one of the few recent publications that explicitly teaches regeneration of embryos and whole plants from isolated grapevine protoplasts, and teaches a medium for regeneration that encompasses the instantly claimed limitations. It would be obvious to combine the teaches for the purpose of successfully regenerating gene edited plants that from the gene edited protoplasts taught by Osakabe.
Closest Prior Art
Claims 13, 15, and 18 appear to be free of the art.
Regarding claims 13, 15, and 18, the closest prior art is found in Van Damme et al. (US Patent No. 10501754), herein referred to as Van Damme
The instant specification describes SEQ ID NOs: 1-4 are mutated DMR6 genes from grapevine (Vitis vinifera) and potato (Solanum tuberosum) (p. 10, lines 31-35, and p. 13, lines 9-12). Van Damme teaches mutations in DMR6 genes to achieve fungal resistance (claim 1 of Van Damme) in potato, specifically teaching a DMR6 nucleotide sequence of SEQ ID NO: 113 which has 98.9% identity (i.e. 1 indel and 1 gap) to instant SEQ ID NO: 4 (claim 1 of Van Damme). Van Damme also teaches identification of the DMR6 coding sequence of Vitis Vinifera (Col. 15, lines 29-48, Table 1).
However, Van Damme does not disclose, teach, or otherwise render obvious one or more mutated sequences selected from the group consisting of SEQ ID NOs: 1-4. The specific mutated sequences of instant SEQ ID NOs: 1-4 are not expressly taught in the prior art and are otherwise not obvious variants.
Response to Arguments
Applicant argues beginning on p. 5 of remarks dated 06/11/2025 the
following arguments:
Osakabe does not disclose or suggest using 0.4 M mannitol in a wash solution. For at least this reason, the claims define over Osakabe alone. Further, Bertini is likewise silent, thus the claims define over the combination of Osakabe and Bertini.
This argument has been fully considered and is found not persuasive for
the following reason(s):
The rejections under 35 U.S.C. § 102 have been withdrawn in view of Applicant’s amendments to claim 1. However, a modified rejection under 35 U.S.C. § 103 has been made in view of the currently amended claims. In the modified rejection, the Tricoli(b) reference teaches the importance of osmotically adjusting medium to prevent protoplast explosion and implosion. Tricoli(b) specifically teaches isolating grapevine protoplasts and washing the protoplast with a medium comprising 0.4 M Mannitol. For this reason, Applicant’s argument is not found persuasive (see 103 rejection above).
Applicant argues beginning on p. 5 of remarks dated 06/11/2025 the following arguments:
None of the cited art discloses or suggests a genome-edited grapevine or potato plant having in its genome one or more mutated sequences selected from SEQ ID NOS: 1-4 as recited in claims 15-18. More specifically and as disclosed of page 12 lines 1 to 3 of the present application, homozygous gene editing of the DMR6 gene was confirmed. The observed mutations were a homozygous deletion of lbp or 2bp, as well as a homozygous insertion of 1 bp (SEQ ID NOs: 1, 2 or 3, respectively). Furthermore, a deletion in DMR6 of 1 bp, which has shown heterozygous editing is represented by SEQ ID NO:4. None of the cited art discloses these mutants in grapevine. For at least this additional reason those claims as amended define over the cited art of record.
This argument has been fully considered and is found not persuasive for
the following reason(s):
The argument is moot because these limitations are not required by the independent claim 1, and are only required by dependent claims 13, 15, and 18 (see Claim Objections and Closest Prior Art sections above).
Applicant argues beginning on p. 6 of remarks dated 06/11/2025 the following arguments:
With regard to the obviousness rejections, Osakabe discloses the genome editing in apple and grapevine protoplasts using CRISPR Cas and micro callus formation (i.e. not producing embryos). Osakabe discloses specifically on page 2859 that "the generation of transgenic plants from these microcalli will require optimization of the combination of hormones and conditions to successfully induce shoot and root formation". In other words, Osakabe is not able to generate embryos or whole plants based on the method disclosed. Osakabe is also silent about the recited mannitol used in the wash solution, as recited in present claims, and Osakabe uses high concentrations of 0.5 to 0.8M mannitol in other media (resuspension media, and enzyme mixtures).
Thus, a difference between Osakabe and the method of claim 1 is the enzyme mixture being used during digestion, using significantly less and different combinations of enzymes and hormones. For example, Osakabe enzyme mix B does not include hemicellulose, and uses less macerozyme. Furthermore, Osakabe is silent about how to generate genetically edited embryos from protoplasts and even more so to generate whole plants and to include a culture media having charcoal, auxins and cytokines.
The effect of the above identified differences is that the method enables the generation of genome edited whole plants with increased efficiency and success, instead of genome edited plant derived microcallus only. The method provides a non-Agrobacterium- mediated, non-integrating gene editing method for further development of non-chimeric gene edited clonally propagated grapevine and potato crops. It was found that a certain media and steps need to be used to successfully obtain embryos and to generate whole plants. As disclosed on page 3 lines 17 to 21 of the present application as filed and supported by the examples (and Figures 4-5), the claimed method generates embryos and eventually germinates into whole genome edited plants. Page 5 lines 10 to 16 of the present application discloses that the concentration and ratio in auxins and cytokines was modified to obtain the most optimal ratio to promote both cell division and cell differentiation and provide for successful regeneration of genome edited whole plants from protoplast. Outside the ratio, there is just proliferation of microcallus and no embryogenesis occurs. Figure 1 of the present application supports the enzymatic digestion resulting in the embryogenic callus. Successful plant development was obtained within 3 to 4 months from the start of the experiment (Figure 2 of the present application). Finally, Example 1 shows that the method of present invention uses lOx more callus (see example 1 of the application) than Osakabe (using only 0.lg) which would not even be feasible to generate whole plants from such little callus as starting material.
Furthermore, as indicated on page 4, lines 22 to 33 of the present application, the control of osmolarity in the method of present invention is an essential feature as governed by mannitol. To obtain a fast and efficient cellular division, it is essential that the protoplasts are not subjected to harsh conditions, i.e. the mannitol should not exceed the recited mannitol concentration, since it will result in non-healthy, suboptimal embryogenic callus protoplasts. The at most 0.4 M mannitol has been found to be effective in the case of grapevine embryogenic callus protoplasts (Figure 1). As noted above, Obasake does not disclose this claimed feature.
This argument has been fully considered and is found not persuasive for
the following reason(s):
A modified rejection under 35 U.S.C. § 103 has been made in view of the currently amended claims. In the modified rejection, Bertini teaches regeneration of plants from grapevine protoplasts and Tricoli(b) teaches the importance of osmotically adjusting medium to prevent protoplast explosion and implosion and teaches washing the protoplasts in a medium comprising 0.4 M Mannitol. Additionally, Eriksson teaches all of the claimed enzymes are commonly employed in protoplast isolation, and concentrations should be adjusted according to the source of tissue. It would therefore be obvious to combine the method of genome-edited protoplast and micro callus taught by Osakabe with the claimed 0.4 M Mannitol taught by Tricoli(b), the enzymes at optimized concentrations taught by Eriksson, and then regenerating the microcallus with the medium taught be Bertini to arrive at the instantly claimed method for the purpose of gene editing protoplasts and regenerating gene-edited grapevine plants (see 103 rejection above). For this reason, Applicant’s argument is not found persuasive.
Applicant argues beginning on p. 7 of remarks dated 06/11/2025 the following arguments:
Bertini discloses the regeneration of plants from embryogenic callus derived protoplast from grapevine. However, Bertini discloses a different and less optimized enzyme mixture comprised of 2% w/v Cellulase Onozuka, 1% w/v Macerozyme R-10, 0.05% w/v Pectolyase Y-23, 10 mM CaCl2, 5 mM 2-(N-morpholino) ethanesulfonic acid (MBS) and 0.5 M mannitol (pH 5.7). This enzyme mixture is suboptimal for both efficiency and cost effectiveness, as is the concentration of mannitol used. Bertini makes use of significantly higher amounts of enzymes including the Pectolyase Y-23 which is one of the most expensive cell wall degrading enzymes in the market. In contrast, the method of present invention uses different combination and a significant lower amount of enzymes and mannitol providing a more efficient and more economical method for the generation of genome edited protoplasts from clonally propagated plant tissue for generation of whole plants.
Furthermore, Bertini discloses different concentrations and combinations of cytokines and auxins (see page 241) and much more activated charcoal, which would teach away from the method of present invention claim 1. Bertini also uses high mannitol which is also outside the claimed range and suboptimal in view of control of osmolarity and to obtain a fast and efficient cellular division. Finally, no gradual decrease in osmolarity is reported in Bertini, and this practice enhances the efficiency of the process of embryogenesis.
Consulting Bertini, starting from Osakabe, would not have guided the skilled person towards the method of claim 1, since there is no evidence that Osakabe can lead to regeneration, nor that Bertini can regenerate from transfected protoplasts. Accordingly, the claims define over the combination of Osakabe and Bertini.
This argument has been fully considered and is found not persuasive for
the following reason(s):
In view of the currently amended claims, Bertini is relied upon for regeneration medium, not enzyme mixture or Mannitol molarity. For this reason, arguments regarding the enzyme mixture and Mannitol molarity are moot. With regard to the argument that Bertini discloses different concentrations and combinations of cytokines and auxins (see page 241) and much more activated charcoal, which would teach away from the method of present invention claim, this argument is not persuasive because Bertini explicitly teaches the protoplasts were cultured for somatic embryogenesis on media comprising 2 mg/L NAA and 0.5 mg/L 6-BAP (p. 241, see protoplast culture for somatic embryogenesis section). Therefore, Bertini teaches a ratio of 4:1 NAA (auxin) to 6-BAP (cytokinin), which encompasses the claimed ratio of auxin:cytokinin in the culture medium being at least about 1:1. Additionally, 2 mg/L NAA is equivalent to 10.7 uM and 0.5 mg/L equivalent to 2.2 uM, and therefore the medium taught be Bertini teaches the claimed medium that comprises between 1.8 to 2.6 uM 6-Benzyladenine (6-BAP), and between 2.5 to 11.5 uM 1- Naphthaleneacetic acid (NAA). Because the instant claims recite the activated charcoal is an optional media component, Bertini is only required to teach the media comprises 1.8 to 2.6 pM 6-Benzyladenine (6-BAP), and between 2.5 to 11.5 pM 1-Naphthaleneacetic acid (NAA). For these reasons, Bertini does in fact teach the claimed medium for regenerating embryos and plants from the microcallus. It would therefore be obvious to combine the gene editing of grapevine protoplast method taught by Osakabe with the grapevine protoplast regeneration method taught by Bertini for the purpose of regenerating gene-edited grapevine plants from protoplasts. For this reason, Applicant’s argument is not found persuasive (see 103 rejection above).
Applicant argues beginning on p. 8 of remarks dated 06/11/2025 the following arguments:
Xu and Tricoli are silent about the generation of whole genome edited plants from the mini-callus colonies by culturing the mini-callus colonies, as recited in step f of amended claim 1. Furthermore, neither discloses a genome edited grapevine or potato plant comprising one or more mutated sequences selected SEQ ID NOS: 1-4.
Furthermore, Xu is silent about grape or potato specifically and relates to round wormwood, which is completely different, and it is difficult to envision why a skilled person working with grape or potato would even consult Xu, without using hindsight in view of the present invention. This document uses a very different culture media including 2.4 dichlorophenoxyacetic acid and 0.65 M mannitol, which is well above the at most 0.4M claimed range used in the method of present invention (with no suggestion that it could be lowered). As indicated mannitol should not exceed the recited mannitol concentration, since it will result in non- healthy, suboptimal embryogenic callus protoplasts. Therefore, only by hindsight would this document be consulted and then used to select one specific feature (of the exact enzyme composition) as recited in the method of present invention, without any incentive for the skilled in the art to do so (and thus leaving out the remaining teachings of the method disclosed in Xu). For example, Tables 2 and 3 of Xu teach the use of 0.65 M being most optimal, which teaches away from the method of present claimed invention.
Tricoli also discloses the use of a significantly different method, disclosing digestion via vacuum infiltration and using pectinase in the enzyme composition, no auxins or cytokinins are disclosed or a step of forming mini-callus to embryo Tricoli discloses the wash solution may include mannitol 0.4 to 0.6M, which values of this range exceed the claimed maximum in the claimed method. Therefore, also Tricoli differs significantly in view of the method of present invention and features need to be selected, without consideration of any other teaching of the reference, from this disclosure to arrive at the claimed subject matter. Such an isolated and uniformed selection would not be performed by the skilled artisan.
In conclusion, no disclosure or hint is found in the cited art which would guide the skilled person to the combination of method steps including the combination of enzyme composition within the recited ranges, wash solution comprising at most 0.4 M mannitol and culturing step using culture medium comprised of auxins, cytokinins, and about | g/L activated charcoal for culturing the mini-callus colonies to form into embryos and eventually to germinate into whole plants. Multiple selections from several documents need to be made and combined without any guidance in the art to do so for the skilled person. Only by using hindsight such selections may be made by the skilled person to arrive at the claimed subject matter. Accordingly, the rejections should be withdrawn.
This argument has been fully considered and is found not persuasive for
the following reason(s):
In view of the currently amended claims, the argument regarding Xu is moot because the 103 rejection does not rely on the Xu reference. Additionally, Tricoli(b) is relied on for teaching isolating grapevine protoplasts and washing the protoplast with a medium comprising 0.4 M Mannitol, not regeneration. As described previously herein, arguments regarding the recited SEQ IDs are moot because only objected claims 13, 15, and 18 require the recited SEQ IDs. Additionally, 0.4-0.6 M Mannitol encompasses 0.4 M Mannitol and is therefore obvious. Additionally, both Tricoli (PCT Patent Application Publication No. WO-2021081397-A1, used in the previous Office Action) and Tricoli(b) (Tricoli, D. (2019). Grape protoplast isolation and regeneration of plants for use in gene editing technology. 2019 research progress report) specifically use in working examples a wash solution comprising 0.4 M Mannitol. For these reasons, Applicant’s argument is not found persuasive.
In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). From the prior art, one having ordinary skill would have been able to combine the teachings of gene-editing protoplasts taught by Osakabe, optimize known enzymes and concentrations as taught by Eriksson, osmotically adjust the wash solution to 0.4 M Mannitol as taught by Tricoli, and then regenerate the gene-edited protoplasts according to the methods and media taught by Bertini for the purpose of regenerating grapevine plants from gene-edited protoplasts.
Conclusion
Claims 13, 15, and 18 are objected to.
Claims 1, 7-12, and 14 are rejected.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JESSICA N STOCKDALE whose telephone number is (703)756-5395. The examiner can normally be reached M-F 8:30-5:00 CT.
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JESSICA N. STOCKDALE
Examiner
Art Unit 1663
/JESSICA NICOLE STOCKDALE/ Examiner, Art Unit 1663
/CHARLES LOGSDON/ Primary Examiner, Art Unit 1662