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 .
Restriction/Election
In response to the communication received on May 6th, 2026, from Marcos P. Rivas, the election of Group I, claims 1-4, 9 and 33, with traversal, is acknowledged. The Applicant alleges that the pending claims are directed to methods and expression constructs for methods that use the disclosed combination to generate somatic embryos in dicot plants and that the claims are not inventive over PCT Application Publication WO 2021/170787 (“Meng”).
This is not found persuasive because Meng need not teach all features of the claims to teach the common technical feature among the groups. Applicants are reminded that the Examiner is permitted to characterize the linking technical feature, namely a first heterologous sequence encoding a BBM or an RWP-RK domain (RKD) and a second heterologous sequence encoding a LEC or FUS polypeptide, and that this technical feature is not required to exactly mirror particular claims.
For reasons of record in the Office Action mailed on March 6th, 2026, the restriction requirement is deemed proper. The linking technical feature has been taught by Meng. Meng teaches a method for plant genome modification through targeting modification of at least one genomic target sequence by at least one regeneration booster, or a combination thereof [Abstract]. “Regeneration booster” refers to a protein/peptide or a polynucleotide fragment encoding the protein/polypeptide which causes improved plant regeneration [pg. 17, lns. 28-31]. Meng teaches that one sequence encodes a PLT or RKD (i.e., RWP-RK domain) and a further sequence encoding BBM, WUS, WOX, GRF, LEC or variant thereof, are introduced into a plant cell, tissue, or organ of a monocotyledonous or dicotyledonous plant [pg. 5, lns. 11-13 and 22-24; claim 18].
The inventions of each of the groups I-III also involve technical features not required by other groups, for the reasons of record stated on page 4, paragraph 4 of the Office action dated March 6th, 2026 (Restriction/Election Requirement).
The requirement is still deemed proper and is therefore made FINAL.
Priority
Applicant’s claim for the benefit of a prior-filed provisional application no. 63/335,488 filed April 27th, 2022, and PCT application no. PCT/US2023/066179 filed April 25th, 2023, under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Thus, the earliest possible priority for the instant application is April 27th, 2022.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on May 15th, 2026, was considered, initialed, and attached hereto. A signed copy of the list of references cited is included with this Office Action.
Status of Claims
Claims 1-4, 9, 11-14, 19, 22-26, and 30-33 filed October 24th, 2024 are pending.
Claims 11-14, 19, 22-26, and 30-32 are withdrawn.
Claims 1-4, 9, and 33 are examined herein.
Claim Objections
Claims 1-4 are objected to because of the following informalities: Claim 1 recites “a method of producing dicot somatic embryo…a second heterologous nucleotide sequence encoding a leafy cotyledon (LEC) polypeptide or FUSCA3 (FUS3) polypeptide…” It is recommended to amend the claim to recite “a method of producing a dicot somatic embryo…” and “…a FUSCA3 polypeptide…” for consistency and clarity.
Claims 2 similarly recites, …” wherein the first heterologous nucleotide sequence encodes BBM polypeptide and the second heterologous nucleotide sequence encodes LEC2 polypeptide.” It is recommended to change this phrasing to “…encodes a BBM polypeptide…” and “…encodes a LEC polypeptide,” and amend claims 3-4 accordingly. Appropriate correction is required.
Claim Interpretation
Claim 1 recites the term “explant”. As defined in the instant specification, suitable explant sources for somatic embryo induction, which can vary by dicot, include cotyledon, hypocotyl, epicotyl, leaves, stems and roots [¶77].
Claim 1 recites a leafy cotyledon (LEC) polypeptide, while dependent claims 2-4 recite specific LEC1 and LEC2 polypeptides. LEC polypeptide is taken to mean the larger group of master transcription factors, with primary LEC proteins LEC1 and LEC2 falling within the larger group. Thus, any LEC polypeptide may read on the LEC polypeptide of claim 1.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-4, 9, and 33 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Applicant describes:
That the expression construct including coding sequences for BBM1/LEC2 or RKD4/LEC2 or BBM1/FUS3 [Example 21] transformed into various plants improved the percentage of explants with somatic embryo formation.
Applicant does not describe:
That the method as claimed can induce somatic embryo formation in any explant type.
The combination of RKD4 and FUS3 polypeptides in a method of producing a dicot somatic embryo.
That the combination of a first heterologous nucleotide sequence encoding a RKD4 and a second heterologous nucleotide sequence encoding a LEC1 (as in claim 3) can generate somatic embryos in any alfalfa genotype that was found to be otherwise recalcitrant [Table 6; Example 3].
That the combination of a first heterologous nucleotide sequence encoding a BBM1 and a second heterologous nucleotide sequence encoding a LEC1 can generate somatic embryos in cotton [Table 20; Example 21].
The instant disclosure describes creating expression constructs including coding sequences for two morphogenic genes, BBM1 and LEC1, BBM1 and LEC2, RKD4 and LEC1, RKD4 and LEC2, and BBM1 and FUS3. The Applicant describes testing the ability of the various constructs to generate somatic embryos in different genotypes of species, including cotton, Brassica napus, poppy, tomato, cucumber, cucumber, carrots, and alfalfa. Examples 1-23 show the use of cotyledons or hypocotyl tissue as explants for dicot somatic embryo production. The term “explant”, as recited in claim 1, covers a broad genus of plant material, including cotyledon, hypocotyl, epicotyl, leaves, stems and roots, as defined in the instant specification (see claim interpretation detailed above). As is known in the art, regeneration of somatic embryos is highly restricted by several factors. These include developmental age and tissue type.
Meristematic tissues, young leaves, and embryos are responsive to hormonal triggers required to switch from a vegetative to an embryonic state as compared to lignified, mature or senescent tissues, which may be impossible to use. Long, Y. et al. (2022. “New Insights Into Tissue Culture Plant-Regeneration Mechanisms.” Front Plant Sci. 13:926752. doi: 10.3389/fpls.2022.926752) teaches that among the explants used in tissue culture, the most widely used are immature embryos or immature cotyledons and hypocotyl segments excised from seedlings [pg. 4, col. 2, ¶2]. Explants in the juvenile-development phase are more regenerative and possess higher totipotency than those of adult explants. For example, a study investigating the frequency of embryonic callus induction among different ages of maize seedlings found a higher frequency of embryonic callus induction for seedlings that were between 2- and 6-cm long than for longer seedlings. Further differences in hormones and nutrients in various explants may play a role in regenerative abilities.
The instant disclosure provides only examples of seeds of various species germinated until the seedlings had a 6-10 cm long hypocotyl and segmented expanded cotyledons [pg. 46, ¶169]. The explants were infected with Agrobacterium and transferred to a medium for somatic embryo induction. A sufficient number of species of explant types were not provided to support the broad genus claim of “explant”, as currently claimed. Due to the unpredictability of regeneration as demonstrated in the art, the instant disclosure lacks adequate description to reflect the potential variation in somatic embryo production between explant types. It is not clear that a different explant would be able to support the function of producing a somatic embryo.
The Applicant describes success in somatic embryo formation using combinations of RKD4/LEC2, BBM1/LEC2, and BBM1/FUS3 expressed in dicot cotyledon and hypocotyl explants. However, the Applicant also concedes that that the combination of a first heterologous nucleotide sequence encoding a RKD4 and a second heterologous nucleotide sequence encoding a LEC1 (as in claim 3) cannot generate somatic embryos in recalcitrant alfalfa lines 1 and 2 [Table 6; Example 3]; that the combination of a first heterologous nucleotide sequence encoding a BBM1 and a second heterologous nucleotide sequence encoding a LEC1 (as in claim 1) did not generate somatic embryos in cotton [Table 20; Example 21]; and lastly fails to disclose the combination of RKD4 and FUS3 polypeptides (as in claim 1) in a method of producing any dicot somatic embryo.
Claim 1 broadly claims a method of producing any dicot somatic embryo comprising expressing in a dicot explant a first heterologous nucleotide sequence encoding a BBM or RKD polypeptide and a second heterologous nucleotide sequence encoding any LEC polypeptide or FUS3 polypeptide, and that any combination of the first and second heterologous nucleotide sequences would induce somatic embryo formation in any explant. The instant disclosure fails to provide written description to support the structure of any combination of the sequences as it directly discloses several combinations being ineffective at inducing somatic embryo formation or fails to describe the combination, in the case of RKD and FUS3. The genus of any combination of the sequences encoding the polypeptides is not reduced to function, with the exception of RKD4/LEC2, BBM1/LEC2, and BBM1/FUS3, in the instant disclosure. Thus, it is not clear that the inventor was in possession of the claimed invention at the broad scope claimed and the claims therefore lack written description.
Examiner notes that Applicant may overcome the above rejection by providing the type of explant used in the instant disclosure and specifying the successful polypeptide combinations for somatic embryo formation.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-4, 9, and 33 are rejected under 35 U.S.C. 103 as being unpatentable over Meng, L. et al. “Method for Rapid Genome Modification in Recalcitrant Plants”, International Publication No. WO 2021/170787 A1, published 02/09/2021 (see IDS filed 05/15/2026), in view of Arling, M. et al. “Plant Explant Transformation”, International Publication No. WO 2020/198408 A1 (see IDS filed 05/15/2026), published 01/10/2020, and Mei, Y. et al. “Boosting Homology Directed Repair in Plants”, International Publication No. WO 2022/002989 A1, published 01/06/2022.
Claim 1 recites a method of producing dicot somatic embryo, the method comprising: expressing in dicot explant a first heterologous nucleotide sequence encoding a BBM or a RWP-RK domain (RKD) polypeptide; expressing in the dicot explant a second heterologous nucleotide sequence encoding a leafy cotyledon (LEC) polypeptide or FUSCA3 (FUS3) polypeptide; and inducing somatic embryo formation in the explant.
Claim 2 recites the method of claim 1, wherein the first heterologous nucleotide sequence encodes BBM polypeptide and the second heterologous nucleotide sequence encodes LEC2 polypeptide.
Claim 3 recites the method of claim 1, wherein the first heterologous nucleotide sequence encodes RKD4 polypeptide and the second heterologous nucleotide sequence encodes LEC1 polypeptide.
Claim 4 recites the method of claim 1, wherein the first heterologous nucleotide sequence encodes RKD4 polypeptide and the second heterologous nucleotide sequence encodes LEC2 polypeptide.
Claim 9 recites the method of claim 1, wherein the dicot explant is from a recalcitrant dicot variety.
Claim 33 recites the method of claim 1, wherein the dicot explant is an explant from cotton, alfalfa, Brassica napus, poppy, tomato, cucumber, sunflower, or carrot.
Regarding claim 1, 9, and 33, Meng teaches a method for rapid genome modification in recalcitrant plants using a combination of “regeneration boosters” which are active or expressed in the plant cell [Abstract; pg. 4, lns. 1-5]. Meng teaches that the term “regeneration booster” refers to a protein/peptide, or a polynucleic acid fragment encoding the protein/polypeptide, which causes improved plant regeneration and regulates somatic embryo formation (i.e., a method of producing somatic embryo) [pg. 17, lns. 28-35]. Meng teaches that a nucleic acid sequence encoding a genome modification, genome editing system, or regeneration booster sequence may be “codon optimized” for the codon usage of a plant target cell of interest [pg. 37, lns. 31-35]. If a nucleic acid sequence is expressed in a heterologous system, codon optimization increases the translation efficiency significantly (i.e., heterologous nucleotide sequence) [pg. 38, lns. 1-4].
Meng teaches using one regeneration booster comprising PLT or RKD4 and a further sequence encoding BBM, WUS, WOX, GRF, or LEC, teaching that the regeneration boosters may be used in combination (i.e., the method comprising: expressing a first heterologous nucleotide sequence encoding a BBM or RKD polypeptide; expressing a second heterologous nucleotide sequence encoding a LEC polypeptide or FUS3 polypeptide) [pg. 5, lns. 11-26; claims 6-7]. Meng teaches that the methods may be applied to monocotyledonous or dicotyledonous plants [pg. 39, lns. 4-5], including Gossypium sp. (cotton) and Brassica napus (i.e., expressing in dicot explant; wherein the dicot explant is an explant from cotton or Brassica napus) [pg. 41, ln. 15-19]. Meng teaches that in particular, plants/plant genotypes that are considered recalcitrant to regeneration can be regenerated efficiently (i.e., wherein the dicot explant is from a recalcitrant dicot variety) [pg. 45, lns. 18-19]. Meng provides the example of embryonic callus induction in recalcitrant elite maize line using regeneration boosters for mature embryo regeneration (i.e., inducing somatic embryo formation in the explant) [Fig. 20; Example 1].
Meng does not explicitly teach expressing both a nucleotide sequence encoding a BBM or RKD and expressing a nucleotide sequence encoding a LEC or FUS3 polypeptide in combination for inducing somatic embryo formation in the explant as specifically arranged in the claims, however combining genes such as BBM, RKD, LEC, and FUS3 was well known at the time of filing.
For instance, Arling teaches a method of producing a transgenic dicot plant that contains a heterologous polynucleotide comprising contacting a dicot vegetative plant organ or its composite tissue with a T-DNA containing the heterologous polynucleotide and a morphogenic gene expression cassette [claim 1]. Arling teaches that the term “transformed plant” refers to a plant that comprises a heterologous polynucleotide within its genome and that additional heterologous polynucleotides may be used in the methods [pg. 21, lns. 8-15; pg. 24, lns. 30-33]. Arling teaches that a wide range of explant types may be used in the method and teaches specific media for somatic soybean embryo maturation after plant tissue is transformed (i.e., a method of producing a dicot somatic embryo, the method comprising expressing in a dicot explant a first heterologous polynucleotide sequence; expressing in the dicot explant a second heterologous nucleotide sequence; and inducing somatic embryo formation in the explant) [pg. 67, Example 1].
Arling teaches that the gene cassette comprises a nucleotide sequence encoding a WUS/WOX polypeptide and/or a BBM polypeptide (i.e., expressing in the dicot explant a first heterologous nucleotide sequence encoding a BBM polypeptide) [claim 2]. Arling lists WUS/WOX genes useful in the methods of the disclosure and teaches that other morphogenic genes useful in the disclosure include LEC1 and LEC2 (i.e., expressing in the dicot explant a second heterologous sequence encoding a LEC polypeptide) [pg. 10, lns. 5-9]. Arling teaches that, according to Kwong, R. et al. (2003. “LEAFY COTYLEDON1-LIKE Defines a Class of Regulators Essential for Embryo Development.” The Plant Cell. 15:5-18), the use of promoters to drive expression of an Arabidopsis LEC1, LEC2 or LEC-like gene in expression cassettes is found to increase the frequency of somatic embryo formation and the recovery of transgenic plants [pg. 70, lns. 20-24; pg. 71, lns. 1-2]. As further evidenced by Kwong, genes encoding Arabidopsis LEC proteins, LEC1, LEC2, and FUSCA3, are unique in that they are the only known embryonic regulators required for normal development during both the morphogenesis and maturation phases (i.e., expressing in the dicot explant a second heterologous nucleotide sequence encoding a LEC polypeptide of FUS3 polypeptide) [pg. 5, col. 2, ¶3], indicating their importance in somatic embryogenesis. Arling provides the example of transformation in soybean, a recalcitrant crop1, and additionally teaches that the methods are applicable in Gossypium hirsutum [pg. 23, ln. 15], which is also regarded in the art as a recalcitrant species2 (i.e., wherein the dicot explant is from a recalcitrant dicot variety; wherein the dicot explant is an explant from cotton) [pg. 23, ln. 7].
Further, Mei teaches a method for boosting plant genome modification in plants, wherein modification is achieved with at least one regeneration booster, or a combination thereof, for improved regeneration of plant tissue, organ or a plant from a single plant cell [Abstract]. Mei teaches that the term “regeneration booster” refers to a protein/peptide, or a polynucleic acid fragment encoding the protein/polypeptide, which causes improved plant regeneration and specifically regulates somatic embryo formation (i.e., a method of producing somatic embryo) [pg. 16, lns. 30-35; pg. 17, lns. 4-8]. Mei teaches that the at least one regeneration booster comprises PLT or RKD4 and that a further regeneration booster is selected from BBM, WUS, WOX, GRF, LEC or variant thereof (i.e., the method comprising expressing a first heterologous nucleotide sequence encoding a RKD4; expressing a second heterologous nucleotide sequence encoding a LEC polypeptide) [claims 10-11].
Mei teaches that the regeneration booster comprises at least one first RBP or PLT sequence, or a sequence encoding the same, preferably at least one RBP sequence, or the sequence encoding the same, and the regeneration booster further comprises: (i) at least one further RBP and/or PLT sequence, or the sequence encoding the same, or a variant thereof, (ii) at least one BBM sequence, or the sequence encoding the same, or a variant thereof, (iii) at least one WOX sequence, including WUS1 , WUS2, or WOX5, or the sequence encoding the same, or a variant thereof, (iv) at least one RKD4 or RKD2 sequence, including wheat RKD4, or the sequence encoding the same, or a variant thereof, (v) at least one GRF sequence, including Zea mays GRF5 and Zea mays TOW/GRF1, or the sequence encoding the same, or a variant thereof, and/or (vi) at least one LEC sequence, including LEC1 and LEC2, or the sequence encoding the same, or a variant thereof [claim 15].
Mei teaches transformation of gene editing components in maize immature embryo [Example 4] and maturation of somatic embryos. Although the provided example is in maize, a monocot, Mei teaches that the methods can be applied to monocotyledonous or dicotyledonous plants and that the plant may originate from Glycine max1 and Gossypium sp. (cotton)2 (i.e., a method of producing dicot somatic embryo; wherein the dicot explant is from a recalcitrant dicot variety; wherein the dicot explant is an explant from cotton) [claim 18].
Given that Meng, Arling, and Mei each teach variants of the claimed heterologous nucleotide sequences encoding a BBM, RKD, LEC, or FUS3 polypeptides for somatic embryo formation in a dicot explant, selecting combinations of these genes as claimed in instant claim 1 to express to try to induce somatic embryo formation would have been prima facie obvious to one of ordinary skill in the art at the time of filing of the instant application. Additionally, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to try the combination of peptide used for inducing somatic embryo formation as specifically recited in instant claims 2-4.
At the time of filing, there was a recognized need in the art to develop regeneration methodology for recalcitrant dicot somatic embryos. For example, Meng teaches that recalcitrant plants specifically have been documented to be difficult to transform and/or regenerate [pg. 3, lns. 14-21]. It was an object of the invention of Meng to provide means and methods to achieve rapid modification and regeneration in recalcitrant plant lines through activating proliferation of somatic cells for embryo formation. Similarly, Arling teaches that a limitation of genetic engineering is plants recalcitrant to transformation and regeneration and that there is a need for plant transformation methods permitting a broader range or regenerable plant explant tissues for commercially viable transgenic plants with improved traits [pg. 1, lns. 18-25].
There were a finite number of identified predictable potential solutions to the recognized problem. Mei teaches that the regeneration booster comprises PLT or RKD4 and that a further regeneration booster is selected from BBM, WUS, WOX, GRF, LEC or variant thereof, including LEC1 and LEC2, and that these genes may regulate somatic embryo formation. Arling further teaches that LEC1, LEC2, and FUS3 are good candidates for expression to increase the frequency of somatic embryo formation and the recovery of transgenic plants. One would have been motivated to try combinations of these genes in dicot somatic embryo formation in other recalcitrant species to further regeneration efficacy and enhance commercial crop traits. One of ordinary skill in the art would have good reason to pursue the known potential solutions (combinations of BBM, RKD, LEC, and/or FUS3 polypeptides) with reasonable expectation of success based on the teachings of Meng, Arling, and Mei, which were available at the time of filing of the instant application.
Thus, it would have been prima facie obvious to try a combination of such polypeptides as recited in instant claims 1-4 (in recalcitrant dicot varieties, including cotton, as in claims 9 and 33, respectively) to one of ordinary skill in the art at the time of filing as there were a finite number of identified, predictable potential genes presented in Meng, Arling, and Mei to solve the recognized problem of developing techniques to modify plant genomes and regenerate somatic embryos to improve plant varieties containing desirable traits, particularly those that are recalcitrant and difficult to transform. The problem of regeneration of recalcitrant plants is well documented across the three cited applications, as is somatic embryo formation.
Conclusion
No claims allowed.
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/EMILY K JOHNSON/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
1 Xu H. et al. (2022. “Progress in Soybean Genetic Transformation Over the Last Decade.” Front. Plant Sci. 13:900318. doi: 10.3389/fpls.2022.900318) teaches that compared to other major crops such as rice, soybean is still recalcitrant to genetic transformation, and transgenic soybean production has been hampered by limitations such as low transformation efficiency and genotype specificity, and prolonged and tedious protocols [Abstract].
2 Pathi, K. et al. (2013. “High-frequency regeneration via multiple shoot induction of an elite recalcitrant cotton (Gossypium hirsutum L. cv Narashima) by using embryo apex.” Plant Signal Behav. 8(1):e22763. doi: 10.4161/psb.22763) teaches that cotton represents a recalcitrant species for regeneration protocols [Abstract].
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