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 .
Continued Examination Under 37 CFR 1.114
1. A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on January 8, 2026 has been entered.
Status of objections and rejections
2. Claims 1-3, 6-15, 17-19, and 21-29 is pending. Claims 4, 5, 16 and 20 are cancelled. Claims 27-29 are previously withdrawn from further consideration by the examiner, 37 CFR 1.142(b), as being drawn to a non-elected invention. Accordingly, claims 1-3, 6-15, 17-19, and 21-26 are examined on merits in the instant Office action.
3. The objection to claims 22 is withdrawn in light of amendments to the claims filed in the papers of January 8, 2026 and upon further consideration.
4. The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
5. Amended Claim 1 interpretation: Amending the claim 1 by reciting “wherein the shoot apical meristem is removed 2-7 days, or 3-4 days , after said contacting” fails to limit the scope of part d) which is also directed to removing the shoot apical meristem. This implies the claim still reads on any one of the options recited in step d). In light of this claim interpretation all previous art rejections remain in place for the reasons of record. However, new art rejections are also included in this Office action.
Claim Rejections - 35 USC § 102
6A. Claim(s) 1-3 and 6-15 remain rejected under 35 U.S.C. 102(a)(1) as being anticipated by Schultheiss et al. (US Patent Publication NO. 2015/0074842 A1; Published March 12, 2015) for the reasons of record stated in the Office action mailed in the papers of October 8, 2025.
Schultheiss et al. disclose a method of Agrobacterium mediated transformation of a plant (e.g. soybean) with a recombinant vector comprising a recombinant DNA construct which comprises a heterologous promoter operably linked to a heterologous polynucleotide comprising a coding sequence and wherein said coding sequence is encoding a protein of interest (e.g. hydrophobin protein) to produce resistance against fungal pathogens, and wherein the vector further carries a polynucleotide sequence encoding selection marker (such as AHAS, bar or dsdA genes), the method comprising steps of : a) providing a plant comprising an axillary meristem and a shoot apical meristem, b) wounding at least part of the axillary meristem to produce a wounded axillary meristem region, c) contacting the wounded axillary meristem region with recombinant DNA construct comprising said polynucleotide and/or with Agrobacterium solution carrying said recombinant plasmid having said heterologous polynucleotide under conditions where the heterologous polynucleotide enters wounded axillary meristem region, d) removing the shoot apical meristem or suppressing the growth of the shoot apical meristem at the same time as step b), growing the plant to regenerate at least part of the wounded axillary meristem region to produce a regenerated axillary meristem or shoot, and wherein the axillary meristem is two axillary meristems, the wounded axillary meristem area is two wounded axillary meristem areas, and the regenerated axillary meristem is two regenerated axillary meristems, and wherein the method comprises removing or suppressing the shoot apical meristem at the same time as step b). The reference further discloses selecting transformed tissues by exposing them to selection marker which are encoded by plant expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. The reference also discloses that infected explants are placed on shoot induction medium with selection agents such as glufosinate (a herbicide). The infected epicotyl explants were then placed on a shoot induction medium with selection agents such as imazapyr (for AHAS gene), glufosinate (for bar gene, herbicide resistance), or D-serine (for dsdA gene). The regenerated shoots were subcultured on elongation medium with the selective agent. The reference clearly discloses contacting plant with said selection agent to eliminate or reduce untransformed tissue and wherein the selection agent occurs during or after instant step e) which involves growing the transformed plant to regenerate at least part of the wounded axillary meristem region to produce a regenerated transformed axillary meristem or shoot. The reference further discloses contacting with selection agent comprises adding the selection agent to a medium in which the transformed plant is growing and untransformed plant is unable to grow. Alternatively, the selection agent is applied to wounded axillary meristem regions and/or regenerated axillary meristem. The transformed tissue explants were placed on said selection agent to at least 2-3 weeks. The reference further discloses performing an assay on the transformed tissues, regenerated axillary meristems or a sample of the regenerated axillary meristem to assess for the presence or absence of the transformed cells and the number thereof. The reference further discloses assaying the transformed plants for disease resistance, wherein presence and expression of the transgene was confirmed due to resistance to fungal pathogen(s). The reference also discloses that following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, and copy number. The reference further discloses that expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis. The reference further discloses obtaining transgenic seeds and raising transgenic progenies from regenerated transformed plants, and wherein the transgenic seeds and raising transgenic progenies comprise the transgene of the transformation event. The reference also discloses that infection with said Agrobacterium solution comprises an infection step and an incubation step. The reference further discloses that said infection step is performed for at least 30 to 60 minutes, and the incubation step is for at least 3 to 5 days in dark. The reference further discloses that said transformed plant is derived from 4 to 8 day seedling explants. The reference further discloses that said axillary meristem is a cotyledonary axillary bud and wherein the method also comprises removing cotyledon of the plant prior to removing or suppressing the shoot apical meristem. See in particular, abstract, paragraphs [0074], [0457], [0543], [0445], [0447], [0450], [0505], [0542], [0543], [0549], [0535], [0551], [0531], [0545], [0535]; and examples 1-8, paragraphs [0522- 0570]
Accordingly, Schultheiss et al. anticipated the claimed invention.
6B. Applicant’s arguments: Applicant primarily argues that claim 1 has been amended to include limitations of claim 5, and thus overcomes this rejection (response page 5).
Applicant’s arguments are carefully considered but are deemed to be unpersusaive. See claim 1 interpretation under item 4. It is important to note that amending the claim 1 by reciting “wherein the shoot apical meristem is removed 2-7 days, or 3-4 days , after said contacting” fails to limit the scope of part d) which is also directed to removing the shoot apical meristem. This implies the claim still reads on any one of the options recited in step d) above.
Accordingly, the rejection is maintained.
7A. Claim(s) 1, 3, 6, 12, 13, 14, 17, 18, 19 and 24-26 remain rejected under 35 U.S.C. 102(a)(1) as being anticipated by Chowrira et al. (Transgenic research; 7:265-271, 1998) for the reasons of record stated in the Office action mailed in the papers of October 8, 2025.
Chowrira et al. disclose a method of electroporation mediated transformation of a plant (e.g. pea, a dicot plant, also a bean plant) with a recombinant vector comprising a recombinant DNA construct which comprises a heterologous promoter operably linked to a heterologous polynucleotide comprising a coding sequence and wherein said coding sequence is encoding a protein of interest (e.g. chimeric pea enation mosaic virus (PEMV) coat protein) to produce resistance against pea enation mosaic virus in transgenic pea plants, the method comprising steps of : a) providing a plant comprising an axillary meristem and a shoot apical meristem, b) wounding at least part of the axillary meristem to produce a wounded axillary meristem region, c) contacting the wounded axillary meristem region with recombinant DNA construct comprising said polynucleotide using electroporation and carrying said recombinant plasmid having said heterologous polynucleotide under conditions where the heterologous polynucleotide enters wounded axillary meristem region, d) removing the shoot apical meristem of the shoot apical meristem at the same time as step b), growing the plant to regenerate at least part of the wounded axillary meristem region to produce a regenerated axillary meristem or shoot, and wherein the method comprises removing the shoot apical meristem at the same time as step b). The regenerated shoots were subcultured on elongation medium. The reference further discloses performing an assay on the transformed tissues, regenerated axillary meristems or a sample of the regenerated axillary meristem to assess for the presence or absence of the transformed cells and the number thereof. The reference further discloses assaying the transformed plants for viral disease resistance, wherein presence and expression of the transgene was confirmed due to resistance to viral pathogen(s). The reference also discloses that following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, and copy number. The reference further discloses that expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis. The reference further discloses obtaining transgenic seeds and raising transgenic progenies from regenerated transformed plants, and wherein the transgenic seeds and raising transgenic progenies comprise the transgene of the transformation event. The reference further discloses that said transformed plant is derived from 4 to 8 day seedling/plant explants. The reference further discloses that said axillary meristem is a cotyledonary axillary bud and wherein the method also comprises removing cotyledon of the plant prior to removing the shoot apical meristem. See in particular, abstract, Figures 1-4, table 1; materials and methods and results, discussion.
Accordingly, Chowrira et al. anticipated the claimed invention.
7B. Applicant’s arguments: Applicant primarily argues that claim 1 has been amended to include limitations of claim 5, and thus overcomes this rejection (response page 5).
Applicant’s arguments are carefully considered but are deemed to be unpersusaive. See claim 1 interpretation under item 4. It is important to note that amending the claim 1 by reciting “wherein the shoot apical meristem is removed 2-7 days, or 3-4 days , after said contacting” fails to limit the scope of part d) which is also directed to removing the shoot apical meristem. This implies the claim still reads on any one of the options recited in step d) above.
Accordingly, the rejection is maintained.
8A. Claim(s) 1-3 and 6-15 remain rejected under 35 U.S.C. 102(a)(1) as being anticipated by Schultheib et al. (WIPO, WO 2013/093738 A1, Published June 27, 2013 for the reasons of record stated in the Office action mailed in the papers of October 8, 2025.
Schultheib et al. disclose a method of Agrobacterium mediated transformation of a plant (e.g. soybean, Arabidopsis, dicots) with a recombinant vector comprising a recombinant DNA construct which comprises a heterologous promoter operably linked to a heterologous polynucleotide comprising a coding sequence and wherein said coding sequence is encoding a protein of interest (e.g. CL protein) imparting rust resistance to Arabidopsis and soybean, and wherein the vector further carries a polynucleotide sequence encoding selection marker (such as bar gene), the method comprising steps of : a) providing a plant comprising an axillary meristem and a shoot apical meristem, b) wounding at least part of the axillary meristem to produce a wounded axillary meristem region, c) contacting the wounded axillary meristem region with recombinant DNA construct comprising said polynucleotide and/or with Agrobacterium solution carrying said recombinant plasmid having said heterologous polynucleotide under conditions where the heterologous polynucleotide enters wounded axillary meristem region, d) removing the shoot apical meristem at the same time as step b), growing the plant to regenerate at least part of the wounded axillary meristem region to produce a regenerated axillary meristem or shoot, and wherein the axillary meristem is more than one (reads on 2 etc.) axillary meristems, the wounded axillary meristem area is more than one wounded axillary meristem areas, and the regenerated axillary meristem is two regenerated axillary meristems, and wherein the method comprises removing or suppressing the shoot apical meristem at the same time as step b). The reference further discloses selecting transformed tissues by exposing them to selection marker which are encoded by plant expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. The reference also discloses that infected explants are placed on shoot induction medium with selection agents such as glufosinate (a herbicide). The infected epicotyl explants were then placed on a shoot induction medium with selection agents such as imazapyr (for AHAS gene), glufosinate (for bar gene, herbicide resistance), or D-serine (for dsdA gene). The regenerated shoots were subcultured on elongation medium with the selective agent. The reference clearly discloses contacting plant with said selection agent to eliminate or reduce untransformed tissue and wherein the selection agent occurs during or after instant step e) which involves growing the transformed plant to regenerate at least part of the wounded axillary meristem region to produce a regenerated transformed axillary meristem or shoot. The reference further discloses contacting with selection agent comprises adding the selection agent to a medium in which the transformed plant is growing and untransformed plant is unable to grow. Alternatively, the selection agent is applied to wounded axillary meristem regions and/or regenerated axillary meristem. The transformed tissue explants were placed on said selection agent to at least 2-3 weeks. The reference further discloses performing an assay on the transformed tissues, regenerated axillary meristems or a sample of the regenerated axillary meristem to assess for the presence or absence of the transformed cells and the number thereof. The reference further discloses assaying the transformed plants for disease resistance, wherein presence and expression of the transgene was confirmed due to resistance to rust disease. The reference also discloses that following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, and copy number The reference further discloses that expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis. The reference further discloses obtaining transgenic seeds and raising transgenic progenies from regenerated transformed plants, and wherein the transgenic seeds and raising transgenic progenies comprise the transgene of the transformation event. The reference also discloses that infection with said Agrobacterium solution comprises an infection step and an incubation step. The reference further discloses that said infection step is performed for at least 30 to 60 minutes, and the incubation step is for at least 3 to 5 days in dark. The reference further discloses that said transformed plant is derived from 4 to 8 day seedling explants. The reference further discloses that said axillary meristem is a cotyledonary axillary bud and wherein the method also comprises removing cotyledon of the plant prior to removing the shoot apical meristem. See in particular, abstract, pages 1-22, Figures 1-11; Tables 1-3; examples 1-11, pages 23-37.
Accordingly, Schultheib et al. anticipated the claimed invention.
8B. Applicant’s arguments: Applicant primarily argues that claim 1 has been amended to include limitations of claim 5, and thus overcomes this rejection (response pages 5-6).
Applicant’s arguments are carefully considered but are deemed to be unpersusaive. See claim 1 interpretation under item 4. It is important to note that amending the claim 1 by reciting “wherein the shoot apical meristem is removed 2-7 days, or 3-4 days , after said contacting” fails to limit the scope of part d) which is also directed to removing the shoot apical meristem. This implies the claim still reads on any one of the options recited in step d) above.
Accordingly, the rejection is maintained.
Claim Rejections - 35 USC § 103
9A. Claim(s) 1-3 and 6-15 remain rejected under 35 U.S.C. 103 as being unpatentable over Schultheiss et al. (US Patent Publication NO. 2015/0074842 A1; Published March 12, 2015), and further in view of Jiang et al. (US Patent Publication No. 2007/0033671 A1, Published February 8, 2007) for the reasons of record stated in the Office action mailed in the papers of October 8, 2025.
Schultheiss et al. teach a method of Agrobacterium mediated transformation of a plant (e.g. soybean) with a recombinant vector comprising a recombinant DNA construct which comprises a heterologous promoter operably linked to a heterologous polynucleotide comprising a coding sequence and wherein said coding sequence is encoding a protein of interest (hydrophobin protein) to produce resistance against fungal pathogens, the method comprising steps of : a) providing a plant comprising an axillary meristem and a shoot apical meristem, b) wounding at least part of the axillary meristem to produce a wounded axillary meristem region, c) contacting the wounded axillary meristem region with recombinant DNA construct comprising said polynucleotide and/or with Agrobacterium solution carrying said recombinant plasmid having said heterologous polynucleotide under conditions where the heterologous polynucleotide enters wounded axillary meristem region, d) removing the shoot apical meristem or suppressing the growth of the shoot apical meristem at the same time as step b), growing the plant to regenerate at least part of the wounded axillary meristem region to produce a regenerated axillary meristem or shoot, and wherein the axillary meristem is two axillary meristems, the wounded axillary meristem area is two wounded axillary meristem areas, and the regenerated axillary meristem is two regenerated axillary meristems, and wherein the method comprises removing or suppressing the shoot apical meristem at the same time as step b). See in particular, abstract, paragraphs [0074], [0457], [0543], [0445], [0447], [0543], [0549], [0535], [0551], [0531], [0545], [0535], examples 1-8, paragraphs [0522- 0570]
Schultheiss et al. do not specifically teach removing or suppressing the shoot apical meristem after instant step c)
Jiang et al. teach a method of plant transformation using polynucleotides and/or polynucleotides encoding polypeptides to transform plants to produce transgenic plants with desired properties. For example, Jiang et al. teach modifying expression of G47 transcription factor to reduce apical dominance. The overexpression of G47 polypeptide produced substantial delay in flowering time and caused a marked change in shoot architecture. The flowering in transgenic plant overexpressing G47 polypeptide was delayed by more than a week than wild type control. The inflorescence of the transgenic plants appeared thick with reduced apical dominance. Likewise transgenic plants
homozygous for a T-DNA insertion in the G438 sequence were obtained and these plants exhibited failure in the development of all types of apical meristem. The reference clearly teach suppressing or reducing removing the shoot apical meristem after plant transformation which is after instant step c). Jiang et al. further teach removing or suppressing the shoot apical meristem 5 days after co-cultivation of Agrobacterium suspension carrying recombinant vector for transformation which reads on instant limitation “shoot meristem is removed or suppressed 2-7 days, optionally 3-4 days after contacting” (see claim 4). Jiang et al. also teach suppressing shoot apical meristem is beneficial to produce new and interesting plant varieties in horticulture industry. See in particular, abstract, paragraphs [0363], [0526], [0600], [0081] [0552].
It would have been obvious and within the scope of an ordinary skill in the art prior to earliest filing date of the claimed invention to modify the method of Schultheiss et al. to include suppression of shoot apical meristem as taught by Jiang et al. for the purpose of creating new plant varieties of potential interest to the ornamental horticulture industry as asserted by Jiang et al. and further discussed above.
9B. Applicant’s arguments: Applicant argues that Scultheiss et al. and Jang et al. cannot be combined because Jang et al. apical meristem suppressing genes or removing apical meristem after contacting with Agrobacterium solution carrying transgene cannot function in soybean. Applicant arguments fails to provide any scientific evidence to support this argument.
Accordingly, the rejection is maintained.
10A. Claim(s) 1 and 15 remain rejected under 35 U.S.C. 103 as being unpatentable over Schultheiss et al. (US Patent Publication NO. 2015/0074842 A1; Published March 12, 2015), and further in view of Chen et al. (Horticulture Research, 5(13): 1-12, published 2018 for the reasons of record stated in the Office action mailed in the papers of October 10, 2025.
Schultheiss et al. teachings are discussed as supra.
Schultheiss et al. do not teach that heterologous polynucleotide comprises one or polynucleotides encoding a Cas protein and/or a guide RNA.
Chen et al. teach using RNA-guided genome editing using CRISPR/Cas9 based system in targeting an endogenous plant gene to suppress or eliminate its expression in plant cells using Agrobacterium mediated plant transformation but without use of selective marker selection of transformed plants. The reference clearly suggests the advantages and precision of using CRISPR/Cas9 based system of targeting endogenous plant genes from diverse plant species to eliminate or suppress expression or generate desired mutants of said targeted endogenous gene. Chen et al. further teach that CRISPR-Cas9 system can be used as an efficient and powerful tool for gene editing and precise genome editing in plants by using multiple guided RNAs (gRNAs) with a 20-22 nt region designed to pair with distinct genomic sites which are followed by the protospacer-adjacent motif (PAM). Chen et al. clearly suggest that using multiple guided RNAs (gRNAs) to achieve multiple edits within the targeted endogenous gene of the plant cell. See in particular, abstract; Figures 1-4; Tables 1-4; results and discussion, methods, pages 1-12.
Given Chen et al. teach that CRISPR/Cas based system of targeting endogenous plant genes can be efficiently and precisely used in regulating plant gene expression by modifying sequence of an endogenous plant gene and eliminates the use of selection marker to obtain transgenic plants, it would have been obvious and within the scope of an ordinary skill in the art prior to earliest filing date instantly claimed invention to have modified expression vector and its recombinant expression cassette of Schultheiss et al. by either substituting its heterologous polynucleotide with an another polynucleotide sequence encoding Cas9 and guide RNA based gene products of CRISPR/Cas9 system to specifically target endogenous gene of Schultheiss et al. plant(s) to regulate its expression to obtain a desirable phenotype of the transgenic plant with a reasonable expectation of success and without any surprising results.
10B. Applicant’s arguments: Applicant primarily argues that claim 1 has been amended to include limitations of claim 5, and thus overcomes this rejection (response page 6).
Applicant’s arguments are carefully considered but are deemed to be unpersusaive. See claim 1 interpretation under item 4. It is important to note that amending the claim 1 by reciting “wherein the shoot apical meristem is removed 2-7 days, or 3-4 days , after said contacting” fails to limit the scope of part d) which is also directed to removing the shoot apical meristem. This implies the claim still reads on any one of the options recited in step d) above.
Accordingly, the rejection is maintained.
11. Claim(s) 1-3 and 6-15 are rejected under 35 U.S.C. 103 as being unpatentable over Schultheiss et al. (US Patent Publication NO. 2015/0074842 A1; Published March 12, 2015), and further in view of Cline (American Journal of Botany 84: 1064–1069, 1997).
Schultheiss et al. teach a method of Agrobacterium mediated transformation of a plant (e.g. soybean) with a recombinant vector comprising a recombinant DNA construct which comprises a heterologous promoter operably linked to a heterologous polynucleotide comprising a coding sequence and wherein said coding sequence is encoding a protein of interest (hydrophobin protein) to produce resistance against fungal pathogens, the method comprising steps of : a) providing a plant comprising an axillary meristem and a shoot apical meristem, b) wounding at least part of the axillary meristem to produce a wounded axillary meristem region, c) contacting the wounded axillary meristem region with recombinant DNA construct comprising said polynucleotide and/or with Agrobacterium solution carrying said recombinant plasmid having said heterologous polynucleotide under conditions where the heterologous polynucleotide enters wounded axillary meristem region, d) removing the shoot apical meristem or suppressing the growth of the shoot apical meristem at the same time as step b), growing the plant to regenerate at least part of the wounded axillary meristem region to produce a regenerated axillary meristem or shoot, and wherein the axillary meristem is two axillary meristems, the wounded axillary meristem area is two wounded axillary meristem areas, and the regenerated axillary meristem is two regenerated axillary meristems, and wherein the method comprises removing or suppressing the shoot apical meristem at the same time as step b). See in particular, abstract, paragraphs [0074], [0457], [0543], [0445], [0447], [0543], [0549], [0535], [0551], [0531], [0545], [0535], examples 1-8, paragraphs [0522- 0570]
Schultheiss et al. do not specifically teach removing the shoot apical meristem after2-7 days or 3-4 days after contacting or after instant step c).
Cline teaches that removal of the shoot apical meristem releases axillary meristems from apical dominance and promotes axillary meristem growth and regeneration (pages 1066–1068), and that the timing of shoot apical meristem removal affects axillary meristem activation (page 1068).
It would have been obvious to one of ordinary skill in the art to select a time for removal of the shoot apical meristem within a known post-transformation recovery period, such as 2–7 days after contacting, as a matter of routine optimization to balance tissue recovery and axillary meristem activation. The claimed time period represents the optimization of a result-effective variable, and no evidence has been provided that the claimed range produces unexpected results relative to the prior art.
12. Claim(s) 1-3 and 6-15 are rejected under 35 U.S.C. 103 as being unpatentable over Schultheib et al. (WIPO, WO 2013/093738 A1, Published June 27, 2013, and further in view of Cline (American Journal of Botany 84: 1064–1069, 1997).
Schultheib et al. teach a method of Agrobacterium mediated transformation of a plant (e.g. soybean, Arabidopsis, dicots) with a recombinant vector comprising a recombinant DNA construct which comprises a heterologous promoter operably linked to a heterologous polynucleotide comprising a coding sequence and wherein said coding sequence is encoding a protein of interest (e.g. CL protein) imparting rust resistance to Arabidopsis and soybean, and wherein the vector further carries a polynucleotide sequence encoding selection marker (such as bar gene), the method comprising steps of : a) providing a plant comprising an axillary meristem and a shoot apical meristem, b) wounding at least part of the axillary meristem to produce a wounded axillary meristem region, c) contacting the wounded axillary meristem region with recombinant DNA construct comprising said polynucleotide and/or with Agrobacterium solution carrying said recombinant plasmid having said heterologous polynucleotide under conditions where the heterologous polynucleotide enters wounded axillary meristem region, d) removing the shoot apical meristem at the same time as step b), growing the plant to regenerate at least part of the wounded axillary meristem region to produce a regenerated axillary meristem or shoot, and wherein the axillary meristem is more than one (reads on 2 etc.) axillary meristems, the wounded axillary meristem area is more than one wounded axillary meristem areas, and the regenerated axillary meristem is two regenerated axillary meristems, and wherein the method comprises removing or suppressing the shoot apical meristem at the same time as step b). The reference further teaches selecting transformed tissues by exposing them to selection marker which are encoded by plant expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. The reference also teaches that infected explants are placed on shoot induction medium with selection agents such as glufosinate (a herbicide). The infected epicotyl explants were then placed on a shoot induction medium with selection agents such as imazapyr (for AHAS gene), glufosinate (for bar gene, herbicide resistance), or D-serine (for dsdA gene). The regenerated shoots were subcultured on elongation medium with the selective agent. The reference clearly teaches contacting plant with said selection agent to eliminate or reduce untransformed tissue and wherein the selection agent occurs during or after instant step e) which involves growing the transformed plant to regenerate at least part of the wounded axillary meristem region to produce a regenerated transformed axillary meristem or shoot. The reference further teaches contacting with selection agent comprises adding the selection agent to a medium in which the transformed plant is growing and untransformed plant is unable to grow. Alternatively, the selection agent is applied to wounded axillary meristem regions and/or regenerated axillary meristem. The transformed tissue explants were placed on said selection agent to at least 2-3 weeks. The reference further teaches performing an assay on the transformed tissues, regenerated axillary meristems or a sample of the regenerated axillary meristem to assess for the presence or absence of the transformed cells and the number thereof. The reference further teaches assaying the transformed plants for disease resistance, wherein presence and expression of the transgene was confirmed due to resistance to rust disease. The reference also teaches that following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, and copy number The reference further teaches that expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis. The reference further teaches obtaining transgenic seeds and raising transgenic progenies from regenerated transformed plants, and wherein the transgenic seeds and raising transgenic progenies comprise the transgene of the transformation event. The reference also teaches that infection with said Agrobacterium solution comprises an infection step and an incubation step. The reference further teaches that said infection step is performed for at least 30 to 60 minutes, and the incubation step is for at least 3 to 5 days in dark. The reference further teaches that said transformed plant is derived from 4 to 8 day seedling explants. The reference further teaches that said axillary meristem is a cotyledonary axillary bud and wherein the method also comprises removing cotyledon of the plant prior to removing the shoot apical meristem. See in particular, abstract, pages 1-22, Figures 1-11; Tables 1-3; examples 1-11, pages 23-37.
Schultheib et al. do not specifically teach removing the shoot apical meristem after2-7 days or 3-4 days after contacting or after instant step c).
Cline teaches that removal of the shoot apical meristem releases axillary meristems from apical dominance and promotes axillary meristem growth and regeneration (pages 1066–1068), and that the timing of shoot apical meristem removal affects axillary meristem activation (page 1068).
It would have been obvious to one of ordinary skill in the art to select a time for removal of the shoot apical meristem within a known post-transformation recovery period, such as 2–7 days after contacting, as a matter of routine optimization to balance tissue recovery and axillary meristem activation. The claimed time period represents the optimization of a result-effective variable, and no evidence has been provided that the claimed range produces unexpected results relative to the prior art.
13. Claim(s) 1, 3, 6, 12, 13, 14, 17, 18, 19 and 24-26 are rejected under 35 U.S.C. 103 as being unpatentable over Chowrira et al. (Transgenic research; 7:265-271, 1998), and further in view of Cline (American Journal of Botany 84: 1064–1069, 1997).
Chowrira et al. teach a method of electroporation mediated transformation of a plant (e.g. pea, a dicot plant, also a bean plant) with a recombinant vector comprising a recombinant DNA construct which comprises a heterologous promoter operably linked to a heterologous polynucleotide comprising a coding sequence and wherein said coding sequence is encoding a protein of interest (e.g. chimeric pea enation mosaic virus (PEMV) coat protein) to produce resistance against pea enation mosaic virus in transgenic pea plants, the method comprising steps of : a) providing a plant comprising an axillary meristem and a shoot apical meristem, b) wounding at least part of the axillary meristem to produce a wounded axillary meristem region, c) contacting the wounded axillary meristem region with recombinant DNA construct comprising said polynucleotide using electroporation and carrying said recombinant plasmid having said heterologous polynucleotide under conditions where the heterologous polynucleotide enters wounded axillary meristem region, d) removing the shoot apical meristem of the shoot apical meristem at the same time as step b), growing the plant to regenerate at least part of the wounded axillary meristem region to produce a regenerated axillary meristem or shoot, and wherein the method comprises removing the shoot apical meristem at the same time as step b). The regenerated shoots were subcultured on elongation medium. The reference further teaches performing an assay on the transformed tissues, regenerated axillary meristems or a sample of the regenerated axillary meristem to assess for the presence or absence of the transformed cells and the number thereof. The reference further teaches assaying the transformed plants for viral disease resistance, wherein presence and expression of the transgene was confirmed due to resistance to viral pathogen(s). The reference also teaches that following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, and copy number. The reference further teaches that expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis. The reference further teaches obtaining transgenic seeds and raising transgenic progenies from regenerated transformed plants, and wherein the transgenic seeds and raising transgenic progenies comprise the transgene of the transformation event. The reference further teaches that said transformed plant is derived from 4 to 8 day seedling/plant explants. The reference further teaches that said axillary meristem is a cotyledonary axillary bud and wherein the method also comprises removing cotyledon of the plant prior to removing the shoot apical meristem. See in particular, abstract, Figures 1-4, table 1; materials and methods and results, discussion.
Chowrira et al. do not specifically teach removing the shoot apical meristem after2-7 days or 3-4 days after contacting or after instant step c).
Cline teaches that removal of the shoot apical meristem releases axillary meristems from apical dominance and promotes axillary meristem growth and regeneration (pages 1066–1068), and that the timing of shoot apical meristem removal affects axillary meristem activation (page 1068).
It would have been obvious to one of ordinary skill in the art to select a time for removal of the shoot apical meristem within a known post-transformation recovery period, such as 2–7 days after contacting, as a matter of routine optimization to balance tissue recovery and axillary meristem activation. The claimed time period represents the optimization of a result-effective variable, and no evidence has been provided that the claimed range produces unexpected results relative to the prior art.
14. Claim(s) 1 and 15 is rejected under 35 U.S.C. 103 as being unpatentable over Schultheiss et al. (US Patent Publication NO. 2015/0074842 A1; Published March 12, 2015), in view of Cline (American Journal of Botany 84: 1064–1069, 1997) and further in view of Chen et al. (Horticulture Research, 5(13): 1-12, published 2018 for the reasons of record stated in the Office action mailed in the papers of April 10, 2025.
Schultheiss et al. teachings are discussed as supra.
Cline teachings are discussed as supra.
Schultheiss et al. do not teach that heterologous polynucleotide comprises one or polynucleotides encoding a Cas protein and/or a guide RNA.
Chen et al. teach using RNA-guided genome editing using CRISPR/Cas9 based system in targeting an endogenous plant gene to suppress or eliminate its expression in plant cells using Agrobacterium mediated plant transformation but without use of selective marker selection of transformed plants. The reference clearly suggests the advantages and precision of using CRISPR/Cas9 based system of targeting endogenous plant genes from diverse plant species to eliminate or suppress expression or generate desired mutants of said targeted endogenous gene. Chen et al. further teach that CRISPR-Cas9 system can be used as an efficient and powerful tool for gene editing and precise genome editing in plants by using multiple guided RNAs (gRNAs) with a 20-22 nt region designed to pair with distinct genomic sites which are followed by the protospacer-adjacent motif (PAM). Chen et al. clearly suggest that using multiple guided RNAs (gRNAs) to achieve multiple edits within the targeted endogenous gene of the plant cell. See in particular, abstract; Figures 1-4; Tables 1-4; results and discussion, methods, pages 1-12.
Given Chen et al. teach that CRISPR/Cas based system of targeting endogenous plant genes can be efficiently and precisely used in regulating plant gene expression by modifying sequence of an endogenous plant gene and eliminates the use of selection marker to obtain transgenic plants, it would have been obvious and within the scope of an ordinary skill in the art prior to earliest filing date instantly claimed invention to have modified expression vector and its recombinant expression cassette of Schultheiss et al. by either substituting its heterologous polynucleotide with an another polynucleotide sequence encoding Cas9 and guide RNA based gene products of CRISPR/Cas9 system to specifically target endogenous gene of Schultheiss et al. plant(s) to regulate its expression to obtain a desirable phenotype of the transgenic plant with a reasonable expectation of success and without any surprising results.
Accordingly, rejection is maintained.
Conclusion
15. Claims 1-3, 6-15, 17-19 and 21-26 remain rejected.
Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Vinod Kumar whose telephone number is (571) 272-4445. The examiner can normally be reached on 8.30 a.m. to 5.00 p.m.
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/VINOD KUMAR/ Primary Examiner, Art Unit 1663