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 .
Continued Examination Under 37 CFR 1.114
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 07/17/2026 has been entered.
Claim Status
Claims 1-3, 5-7 and 13-27 are pending.
Claims 1-3, 5-7 and 13-27 are being examined.
All previous objections and rejections not set forth below have been withdrawn in view of applicant’s amendments to the claims.
Claim Rejections - 35 USC § 103
Claims 1-3, 5-7, 13-20 and 22-27 are rejected under 35 U.S.C. 103 as being unpatentable over Malabarba et al. (New Strategies to Overcome Present CRISPR/Cas9 Limitations in Apple and Pear: Efficient Dechimerization and Base Editing, 2021, Int. J. Mol. Sci., 22:319; first published on 30 Dec. 2020) in view of van der Vyver et al. (In vitro selection of transgenic sugarcane callus utilizing a plant gene encoding a mutant form of acetolactate synthase, 2013, In Vitro Cell. Dev. Biol., Plant, 49:198-206), Ntui et al. (Robust CRISPR/Cas9 mediated genome editing tool for banana and plantain (Musa spp.), 2020, Curent Plant Biology, 21:100128), Tripathi et al. (Efficient regeneration and transformation of plantain cv. “Gonja manjaya” (Musa spp. AAB) using embryogenic cell suspensions, 2012, In Vitro Cell. Dev. Biol., Plant, 48:216-224) and Chen et al. (CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture, 2019, Annu. Rev. Plant Biol. 2019. 70:667–97), and in evidence of Belser et al. (Telomere-to-telomere gapless chromosomes of banana using nanopore sequencing, 2021, Commun. Biol., 4:1047).
Claims 1 and 24 are drawn to a method of generating at least one banana embryo or a plant from an embryogenic cell or protoplast derived from an embryogenic cell suspension comprising at least desired one mutation in at least one endogenous acetolactate synthase (ALS) gene providing resistance to an ALS inhibitor.
Malabarba et al. describes a method using the CRISPR-Cas9 based base editing technique using a Cas9 nickase and cytosine base editor (CBE), which introduces at least one desired C to T mutation, to successfully edit the target gene(s) that include at least one endogenous acetolactate synthase (ALS) gene resulting in ALS inhibitor (chlorsulfuron) resistant plants (abstract; page 3, para 1, line 7-8). Malabarba et al. also teaches successful use of CRISPR/Cas based gene editing in banana (page 2, para 1, line 6-10). Malabarba et al. describes a nCas9 (nickase) fused to a cystine deaminase base editor and gRNAs to edit the endogenous ALS genes (page 6, para 1, line 1-16), as recited in claim 3. It is well-known in the art that the gene editing function of sequence-specific Cas9 endonuclease comprises homology-directed repair (HDR) in conjunction with a donor template, which is often an exogenous DNA encoding the gRNA sequence (Chen et al.; Fig.2b; page 673, para 2.2.2, line 1-8) comprising the desired mutation for the target gene (i.e. PDS gene, in this case), as recited in claims 5-6.
Malabarba et al. describes the nCas9, a cytidine deaminase, and gRNAs (reads on to “agents”) operable to introduce the at least one desired (C to T) mutation in the target ALS sequence encoded by the vector, pDen-Cas9_PmCDA1_UGI developed using the Gateway vector cloning system (Fig. 5) (reads on to “nucleic acid construct”) which also encodes a selectable marker (NPTII gene), as recited in claim 15. The method (base editing using CRISPR-Cas technique) described by Malabarba et al. generates genome-edited plants that does not comprise heterologous DNA in apple and pear (p.2, para 2, line 16-17), as recited in claim 20.
Malabarba et al. teaches Agrobacterium-mediated transformation (page 6, para 1, line 2-3), as recited in claim 7. Malabarba et al. describes that herbicides from the sulfonylurea class comprising chlorsulfuron are ALS inhibitors (page 3, para 1, line 7-8), as recited in claims 18 and 27.
It is known in the art that culturing a mixed population of plant cells that contain transgenic or genome edited herbicide-resistant cells in the presence of the specific herbicide imposes a strong selection pressure resulting in at least 90% (nearing 100%) of the surviving population possessing the mutated (ALS) resistance gene, as recited in claim 19.
However, Malabarba et al. does not describe a banana embryogenic cell or a protoplast derived from an embryogenic cell suspension and generating at least one banana embryo or plant.
van der Vyver et al. describes transforming sugarcane embryogenic callus cell explants (p. 200, left column, para 5-6) and selecting the ALS herbicide (chlorsulfuron) resistant embryos and subsequently ALS herbicide (chlorsulfuron) resistant plants (p.200, right column, para 2, line 1-4).
Tripathi et al. describes creating embryogenic cell suspension using friable embryogenic calli in banana with many translucent proembryos (which ultimately become embryos before regenerating) that were transferred to liquid medium and homogenous cell suspensions were established (page 216, left column, para 1, line 9-11).
Ntui et al. describes a robust CRISPR/Cas9 mediated genome editing tool for banana, Musa spp. (title and abstract) using multiple gRNAs targeting phytoene desaturase (PDS) gene (abstract). It describes regenerating transgenic (genome edited) banana plants (page 5, left column, para 5, line 4-7) after transforming embryogenic cells in an embryogenic cell suspension (page 3, left column, para 7, line 1-2; page 5, left column, para 5, line 1-2), as recited in claims 1-2. Ntui et al. describes regenerating (reads on to “growing”) several banana plants from at least one cell (page 5, left column, para 5, line 4-6), as recited in claims 22-23. Ntui et al. teaches achieving at least 90% (100%) mutation efficiency (which also reads on to “cells in the population comprise mutated ALS”) in at least one banana cultivar (page 8, right column, para 2, line 9-10), as recited in claim 19.
It would have been obvious to an ordinarily skilled artisan to modify the method of editing the ALS gene using CRISPR-Cas technique and select ALS herbicide (chlorsulfuron) resistant banana embryos and subsequently ALS herbicide (chlorsulfuron) resistant banana plants, as described by Malabarba et al. and van der Vyver et al., in embryogenic cells in commercially important banana varieties, as described by Ntui et al.
The ordinarily skilled artisan would have been motivated to edit the endogenous ALS gene in banana with a reasonable expectation of success to develop commercially important banana embryos and/or plants (as recited in claims 1 and 22-24) that are resistant to herbicides from the sulfonylurea class comprising chlorsulfuron, which is also a commercially valuable trait. Harvesting a fruit from the genome edited banana plant (as recited in claim 22) is a routine standard process.
Regarding claims 13-14 and 25, Physically separating the mutated (transgenic or genome edited) cells from non-mutated ones, as recited in claim 13, during subculturing in selection media containing the selection agent, is a standard practice while using (embryogenic) cell suspension culture comprising small clumps of friable callus for transformation, as described by Tripathi et al. Genotyping the mutated cells and/or the genome edited plants to confirm the presence of the desired mutation, as recited in claims 14 and 25, is also a routine and standard practice in the art, as also described by Ntui et al. (page 3, right column, para 3).
Regarding claims 16-17 and 26, Malabarba et al. teaches the ALS sequences from apple (Gene ID: MD06G1028700 and Uniprot Accession No. A0A498JQ03) and pear (Gene ID NO. PCP000109 and Uniprot Accession No. A0A5N5HW78) (page 13, para 2, line 1-2; Fig. 3A). It describes designing the guide RNA (sgRNA) around the coding sequence for the polypeptide “SIPVVAITGQVPRRMIGTDAFQETPIVE” (page 7, Fig. 3A). Identifying homologues, in any plant including banana, based on sequence identity has been a routine and standard practice in the prior art. It would have been obvious to an ordinarily skilled artisan to identify different alleles of the ALS gene (ALS1 and/or ALS2) in one or more commercially important banana species, as recited in claim 16, using the banana genome hub, as described by Ntui et al. (page 2, right column, para 3, line 1-4).
Malabarba et al. describes mutating the ALS genes in apple and pear so that P185 and P192 are mutated to leucine (page 8, para 1, line 4-5), serine (p.6, para 1, line 12-13), cysteine (page 8, para 1, line 6), phenylalanine (page 8, para 1, line 8) and/or arginine (page 8, para 1, line 9-10) resulting in a mutant apple plant resistant to ALS inhibitor chlorsulfuron. The proline residue at 187 in ALS1 and at 181 in ALS2 in banana are aligned to the apple ALS polypeptide at position P185. That corresponding proline residue, as shown in the red box below, is conserved in all the ALS sequences. It would have been obvious to an ordinarily skilled artisan to substitute the corresponding P187 in ALS1 and/or P181 in ALS2 in banana with leucine and/or serine (as recited in claims 17 and 26) with a realistic objective to get a mutant banana plant that is resistant to ALS inhibitor chlorsulfuron.
Banana ALS proteins, ALS1 (Ma06_g18100) and ALS2 (Mal0_gl 1980), as also acknowledged by the Applicant as SEQ ID NOs: 9-10 (Spec, p.53, last 2 lines; Table 3), are known in the prior art as described in the banana genome hub database, which is described by Tripathi et al. (p.5, right column, last para and footnote) and Ntui et al. (p.2, right column, para 6, line 3). All the ALS sequences from apple, pear, and banana (MsALS1 and MsALS2) are aligned in respect to “SIPVVAITGQVPRRMIGTDAFQETPIV E”, as shown below.
CLUSTAL 2.1 multiple sequence alignment
AppleALS MAAVAPPSSSSFSKPSLFSSSAKSHHPISRFNLPFSPLPQKPSPLRHLRISSSLSPKTDT
PearALS MAAIA PPSSYSFSKPSPFSSSAKSHHPISRFNPPFSPLPQKPSPLRHLRIFSSLSPKTDT
Malabarba3A ------------------------------------------------------------
MsALS1 --MASSAAAAVAAAGAVITPSKSSHPPFSAAYRLPFPLPKPLHSLSTRHPHVRPISASAD
MsALS2 --------MASSTPGAAIA PSNPSLWPFSAASRLLVPFPKPLLSLSPSHRNVRPISASAD
AppleALS TITTTTS----NPLDLEAPISRFAPDEPRKGADVLVEALERQGVTDVFAYPGGASMEIHQ
PearALS TITTATTTTTTNPLHPEVPISRFAPDEPRKGADVLVEALERQGVTDVFAYPGGASMEIHQ
Malabarba3A ------------------------------------------------------------
MsALS1 RRQLPSAAAPTADATTAPLLRNFAPDEPRKGADILVEALEREGVTDLFAYPGGASMEIHQ
MsALS2 RHQTPSAAASTAATTAVPLLRNFAPDEPRKGSDILVEALEREGVTDLFAYPGGASMEIHQ
AppleALS ALTRSSSIRNVLPRHEQGGVFAAEGYARASGRPGVCIATSGPGATNLVSGLADALMDSIP 176
PearALS ALTRSSSIRNVLPRHEQGGVFAAEGYARASGRPGVCIATSGPGATNLVSGLADALMDSIP 180
Malabarba3A ---------------------------------------------------------SIP 3
MsALS1 ALTRSPSITNHLLRHEQGEVFAASGYARSTGRPGVCIATSGPGATNLVSGLADALLDSVP 178
MsALS2 ALTRSPSITNHLLRHEQGEIFAASGYARSTGRPGVCIATSGPGATNLVSGFADALLDSVP 172
*:*
[AltContent: rect]AppleALS VVAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDVEDIPRVVREAFFLATSGRPG
PearALS VVAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDVEDIPRVVREAFFLATSGRPG
Malabarba3A VVAITGQVPRRMIGTDAFQETPIVE-----------------------------------
MsALS1 LVAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLNVDDIPRIIKEAFFVATTGRPG
MsALS2 LVAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLNVDDIPRIIKEAFFLASTGRPG
:************************
AppleALS PVLIDIPKDVQQQLVVPNWDQPIKLPGYMSRLPKSPNEGHLEQIVRLVSESKKPVLYVGG
PearALS PVLIDIPKDVQQQLVVPNWDQPIKLPGYMSRLPKSPNEGHLEQIVRLVSESKKPVLYVGG
Malabarba3A ------------------------------------------------------------
MsALS1 PVLVDIPKDIQQQLAIPVWDPPLRLPGYISRLPRLPARPLLDQILRLVSESRRPVLYVGG
MsALS2 PVLVDIPKDIQQQLAIPVWDPPLRLPGYISRLPKPPSRCLLDQIIRLVSESHRPVLYVGG
AppleALS GCLNSSEELRRFVELTGIPVASTLMGLGAYPCNDEELSLQMLGMHGTVYANYAVDKSDLL
PearALS GCLNSSEELRRFVELTGIPVASTLMGLGAYPCNDEELSLQMLGMHGTVYANYAVDKSDLL
Malabarba3A ------------------------------------------------------------
MsALS1 GCLNSSEELHRFADLTGIPIASTLMGLGAYP-TDAELSLKMLGMHGTVYANYAIDKADLL
MsALS2 GCLNSSEELRRFADLTGIPIASTLMGLGVYP-TDAELSLKMLGMHGTVYANYSVDKADLL
AppleALS LAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSVCADVKPALEGLNRILE
PearALS LAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSVCADVKSALEGLNRILE
Malabarba3A ------------------------------------------------------------
MsALS1 LAFGVRFDDRVTGKIEAFASRAKIVHIDIDPAEIGKNKQPHVSICADVKLALQGMNALME
MsALS2 LALGVRFDDRVTGKLEAFASRAKIVHIDIDPAEIGKNKQPHVSLCADVRLALQGMNALME
AppleALS QKESKVKLDFSAWRAELKEEKVKFPLGFKTFGDSISPQNAIQVLDELTDGNAIISTGVGQ
PearALS QKESKVKLDFSAWRAELKEEKVKFPLGFKTFGDSISPQNAIQVLDELTDGNAIISTGVGQ
Malabarba3A ------------------------------------------------------------
MsALS1 ETEIYRKFDFSTWREELDKQKKIYPLNYKTFGDQIPPQYAIQVLDELTNGEAIITTGVGQ
MsALS2 ESGIHQKFDFSTWRKELDQLKKAYPLSYKTFGDLIPPQYAIQVLDELTNGEAIISTGVGQ
AppleALS HQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVANPDSVVVDIDGDGSFLMNVQE
PearALS HQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVANPDSVVIDIDGDGSFLMNVQE
Malabarba3A ------------------------------------------------------------
MsALS1 HQMWAAQYYSYKRARQWLTSAGLGAMGFGLPAAAGAAVGNPGVTVVDIDGDGSFQMNAQE
MsALS2 HQMWAAQYYSYKRARQWLTSGGLGAMGFGLPAAAGAAVGNPGVTVVDIDGDGSFQMNAQE
AppleALS LATISVEKLPVKIMLLNNQHLGMVVQWEDRFYKANRAHTYLGNPANESEIFPNMLKFADA
PearALS LATISVEKLPVKIMLLNNQHLGMVVQWEDRFYKANRAHTYLGNPANESEIFPNMLKFADA
Malabarba3A ------------------------------------------------------------
MsALS1 LAMIRIENLPVKIMVLNNQHLGMVVQWEDRFYHRNRAHTYLGNPANETEVFPDFLKISEG
MsALS2 LAMIRIENLPVKMMVLNNQHLGMVVQWEDRFYRSNRGHTYLGNPANESEIFPDFLKITEA
AppleALS CGIPAARVTKKEDLKAAIQKMLDTPGPYLLDVIVPHQEHVLPMIPSGGAFKDVITEGDGR
PearALS CGIPAARVTKKEDLKAAIQKMLDTPGPYLLDVIVPHQEHVLPMIPSGGAFKDVITEGDGR
Malabarba3A ------------------------------------------------------------
MsALS1 YGIPAARVTKKSEVREAIRKMLETSGPYLLDVIVPHEEHVLPMIPSGGAFKDMILDGDGR
MsALS2 YGIPAARVTKKSEVREAIRKMLKTPGPYLLDVIVPHEEHVLPMIPSGGAFKDMILDGDGR
AppleALS SSY-
PearALS SSY-
Malabarba3A ----
MsALS1 TPY-
MsALS2 TPPH
Claims 21 is rejected under 35 U.S.C. 103 as being unpatentable over Malabarba et al. in view of van der Vyver et al., Ntui et al., Tripathi et al. and Chen et al., as applied to claims 1-3, 5-7, 13-20 and 22-27 above, and further in view of Hu et al. (CRISPR/Cas9-mediated genome editing of MaACO1 (aminocyclopropane-1-carboxylate oxidase 1) promotes the shelf life of banana fruit, 2021, Plant Biotech. J., 19:654–656; published on 28th January 2021).
Malabarba et al. in view of van der Vyver et al., Ntui et al. and Tripathi et al. describes a method of generating at least one banana embryo and/or a banana plant comprising at least one desired mutation in at least one of the endogenous acetolactate synthase (ALS) gene(s) in an embryogenic cell so as to provide resistance to an ALS inhibitor, wherein the embryogenic cell is derived from an embryogenic cell suspension, as discussed above.
However, Malabarba et al. in view of van der Vyver et al., Ntui et al. and Tripathi et al. does not describe gene stacking by mutating ACO, ACS or PPO gene(s) in the banana plant which is already having the mutated ALS gene(s).
Hu et al describes commercially important trait of increased shelf-life of banana by suppressing the production of ethylene by editing the ACO gene using CRISPR-Cas system in banana (MaACO1) (title; page 656, left column, para 4, line 2-4). Gene stacking is a well-known standard technique in the art to introduce multiple desirable genes and/or mutations in a single plant to introduce valuable traits into crop varieties.
It would have been obvious to an ordinarily skilled artisan to edit the endogenous ACO gene(s) in banana, as described by Hu et al., in the same plant having the mutated ALS gene(s), as described by Malabarba et al. in view of Ntui et al. and Tripathi et al. The artisan would have been motivated to do it with a reasonable expectation of success to extend the shelf-life of the already herbicide resistant banana plants comprising ALS mutation(s).
Response to Applicant’s arguments
The argument set forth in the Applicant’s response on 07/17/2026 has been fully considered but is not found persuasive.
All the arguments regarding the rejections under 35 U.S.C. 103 boil down to, “None of the cited references, either alone or in combination, suggest using ALS selection at the embryogenic cell stage in any plant species, let alone banana. Moreover, none teach or suggest using ALS inhibitor resistance as an early-stage proxy for identifying embryogenic material that has also been edited in a separate target gene of interest. Rather, the cited references are focused solely on generating new plant traits” (response, p.8, last para in its entirety).
The Applicant also argues that “Malabarba's teachings are specifically directed to the problem of chimerism that is associated with CRISPR/Cas9 editing in apple and pear” (response, p.10, last para, line 3-4) and “the solution to chimerization that is taught in Malabarba is direct and unambiguous - it teaches that an adventitious regeneration step should be used (response, p.10, last para, line 7-8). Argument continues, “Modifying Malabarba to arrive at the claimed method, which requires ALS selection at the embryogenic cell stage, would therefore undermine Malabarba's central teaching regarding the importance of adventitious regeneration” (response, p.11 para 1, last 3 lines).
The Examiner disagrees. Malabarba et al. teaches editing ALS genes to develop commercially important trait of (ALS) herbicide resistance in apple and pear plants using CRISPR-Cas technique while van der Vyver et al. describes transforming sugarcane embryogenic callus cell explants (p. 200, left column, para 5-6) and selecting the ALS herbicide (chlorsulfuron) resistant embryos and subsequently ALS herbicide (chlorsulfuron) resistant plants (p.200, right column, para 2, line 1-4). On the other hand, Tripathi et al. describes creating embryogenic cell suspension using friable embryogenic calli in banana while Ntui et al. describes a robust CRISPR/Cas9 mediated genome editing in banana (title and abstract). Ntui et al. describes regenerating genome edited banana plants (page 5, left column, para 5, line 4-7) after transforming embryogenic cells in an embryogenic cell suspension (page 3, left column, para 7, line 1-2; page 5, left column, para 5, line 1-2).
The Examiner cited the reference of Malabarba et al. for its teaching of editing plants using CRISPR/Cas9 base editing technique in apple and pear using two easily scorable genes: acetolactate synthase- ALS and PDS (abstract) and not for dechimerization in specific plants. There is no evidence to indicate that the method used in apple and pear would not work in banana in terms of developing specific herbicide resistance in banana by mutating ALS and/or another target gene(s) (e.g., PDS, as described by Malabarba et al.). There is also no reason to believe that the embryogenic callus cell explants (p. 200, left column, para 5-6) and selecting the ALS herbicide (chlorsulfuron) resistant embryos (developed from the genome edited embryogenic callus cell explants) and subsequently ALS herbicide (chlorsulfuron) resistant plants (p.200, right column, para 2, line 1-4), as observed in (monocot) sugarcane and as described by van der Vyver et al., would not work for (monocot) banana.
The Applicant is reminded that the requirement for an explicit analysis for Prima Facie case of obviousness does not require record evidence of an explicit teaching of a motivation to combine in the prior art. Any relevant information and/or (scientific) teaching described in one or more prior art would suffice. See MPEP ¶ 2143.
Conclusion
No claim is allowed.
Communication
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JAY CHATTERJEE whose telephone number is (703)756-1329. The examiner can normally be reached (Mon - Fri) 8.30 am to 5.30 pm..
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Bratislav Stankovic can be reached at (571) 270-0305. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
J.C.
/Jay Chatterjee/ Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/ Supervisory Patent Examiner, Art Units 1661 & 1662
Prosecution Insights