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 .
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 .
Election/Restrictions
Applicant’s election with traverse of group I, consisting of claims 1-2, 4, 6-8, 10, 12, 15, 20, 22, 31, 33 & 38, in the reply filed on 22 June 2026 is acknowledged. Applicant has elected the species of (i) CMS-C, (ii) Female fertile and cytoplasmic male sterility (CMS) male fertile, (iii) SEQ ID NO.5, and (iv) Rf4.
Regarding inventive groups; Applicant urges the prior art does not teach a plant which is a haploid inducer and comprises CMS cytoplasm, used to generate progeny [Remarks, p.1, ¶.3]. Applicant argues that the alternate mutation described in KWS renders the instant application distinct. This is unconvincing, as disruption of cenh3 results in haploid induction characteristics. Functionally equivalent mutation of the previously described gene results in the same phenotype, replicating the same method.
Regarding species elections; Applicant argues (i) that CMS-C, CMS-S and CMS-T are functionally equivalent [Remarks, p.2, ¶.5—p.3, ¶.1], that (ii) both recited states lead to the same technical result of the CHIP being a female parent [p.3, ¶.2], that (iii) there is no functional distinction between SEQ ID NO.5 & 6 because, “other cenh3 knockout mutations…achieve the same functional result of disrupting CENH3 function to enable haploid induction” [p.3, ¶.3], and (iv) the distinct restorer alleles recited function equivalently to restore fertility to a CMS plant [p.3, ¶.4].
With respect to (ii) & (iii); Applicant’s arguments are convincing, and the previous species restrictions of the male fertility status of a CMS line used as a parent, and, restriction between SEQ ID NO.5 and SEQ ID NO.6 are withdrawn.
With respect to (i) & (iv); Applicant’s arguments are unconvincing, as the three CMS systems have different genetic mechanisms and are not equivalent in their resulting phenotypes in plants [See p.77, col.2, ¶.3—p.78, col.2, ¶.1 in Weider Crop Sci. 49:77–84 (2009); Published January 2009]. Each of these CMS systems has a different combination of restorer alleles (i.e. Rf genes) which are not interchangeable or generic in their applied use or ability to modify the fertility of all CMS cytotypes [id]. These different CMS systems are not functionally equivalent with respect to their genetic architectures or pleiotropic effects on plant phenotype or practical utility.
Because of this, the previous restriction of group I to claims 1-2, 4, 6-8, 10, 12, 15, 20, 22, 31, 33 & 38 is maintained. Species election of (i) CMS-C and (iv) Rf4 is maintained. Species of (ii) and (iv) are rejoined for examination.
Claims 34-35, 40-43, 46 & 48 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claim Status
Claims 1-2, 4, 6-8, 10, 12, 15, 20, 22, 31, 33 & 38 are under examination on the merits.
Claims 34-35, 40-43, 46 & 48 are withdrawn as non-elected subject matter.
Claims 3, 5, 9, 11, 13-14, 16-19, 21, 23-30, 32, 36-37, 39, 44-45 & 47 are canceled.
Priority
Claims 1-10 receive the U.S. effective filing date 19 May 2022.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
Claim 12 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 12 recites a series of genome editing enzymes as ‘AsCas12a, LbCas12a…Mb2Cas12a, etc’. The inclusion of ‘et cetera’ in listed claim limitations is indefinite as it provides no indication or definition of what is encompassed by ‘etc’.
Therefore, the claim is considered indefinite because there is a question as to the metes and bounds of the claim limitations, and how one would avoid infringement.
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 1-2, 4, 6-8, 10, 12, 15, 20, 22, 31, 33 & 38 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.
Broadest reasonable interpretation of claim 1, from which all other claims depend, is that it encompasses a method applicable to any and all plants. The claim is not specifically limited to maize, and dependent claims 31 & 38 only narrow this scope by limiting to several crop genera.
Review of the specification indicates Applicant has only described their method specifically in maize, using the known, maize-specific cytoplasmic sterility system based on interaction of CMS-C cytoplasm and the Rf4 restorer locus [Specification, p.26, Example 1; p.30, Example 2; p.33, Example 3; p.42, Example 4; p.46, Example 5].
Applicant provides a background on cytoplasmic male sterility systems, generally, but all examples are specific to maize [id]. All necessary elements of the method, other than editing of the conserved cenh3 gene, are specific to corn. This includes the male sterility systems described (i.e. CMS-C/Rf4), and markers necessary for identification of haploid plants which are R1-Navajo [Ex. 1] and R1-SCM2 [Ex.2].
Prior art directed to this topic indicates that cytoplasmic male sterility systems are specific to crop species, and within species the functionality of various CMS systems is genotype specific. Different genetic backgrounds may have uncharacterized restorer alleles or environmental interactions that render the CMS system ineffective [see p.78, col.2, ¶.3; p.80, col.2, ¶.3 & Figure 2; and, p.81, col.1, ¶.4—p.82, col.2, ¶.2 in Wieder].
Applicant does not describe a method of conferring CMS in any and all plants, or that would reasonably work outside of the specific maize CMS-C/Rf4 system.
The written description does not reasonably convey to one skilled in the relevant art that the inventor had possession of the claimed invention. As such, claim 1 and its dependent claims 2, 4, 6-8, 10, 12, 15, 20, 22, 31, 33 & 38 are rejected.
Claim 1, from which all other claims depend, recites a method of conferring cytoplasmic male sterility (CMS) via transfer of CMS cytoplasm (i.e. CHIP) to another plant (i.e. DIP). The limitations of this claim are drawn to production of a functionally male sterile plant in a different nuclear genetic background (i.e. DIP).
Review of Applicant’s specification describes the mechanism by which CMS factors work in maize, generally [p.26, Example 1; p.30, Example 2; p.33, Example 3; p.42, Example 4; p.46, Example 5]. Applicant’s examples provide description of the generation of haploid-inducing lines with homozygous markers for detection. Applicant’s examples describe crossing of these materials (i.e. CHIPs) to regular maize lines (i.e. DIPs) and regeneration of plants having markers for (i) cenh3 mutation, (ii) linked anthocyanin markers to identify progeny, and (iii) cytoplasm CMS-C type.
Applicant does not indicate that the recovered plants are male sterile. Although molecular markers are described indicating the genotype, no phenotypic data is reported or described validating that the recovered maize plants are male sterile, or that the method described predictably confers male sterility (i.e. CMS). Prior art on the subject of CMS systems indicates their effectiveness often is dependent on genetic background or environmental conditions, and thus requires experimental validation [Weider, p.80, col.2, ¶.3 & Figure 2].
Applicant’s examples stop short of describing whether the plants generated by the proposed method generate pollen or are sterile, instead pointing to their genotypic/marker constitutions [p.32, Tables 3-5]. It is reasonable to consider that a plant may have a particular marker constitution but still fail to be male-sterile. Applicant presents no phenotypic data indicating derived progeny plants are male sterile.
Validation of the phenotypic effect of such CMS-C background transfer is required because CMS can be unstable or not work [Weider, p.80, col.2, ¶.3 & Figure 2]. The instability of various CMS cytotypes is due to the background nuclear genome in which they occur, effects of various restoration factor (Rf) alleles present, and the extent that each background interacts with environmental conditions [id].
Moreover, having obtained plants from the claimed method, it would be reasonably easy for one to grow said plants to maturity and record flowering characteristics. It is unclear why Applicant, having gone to the effort of gene editing, backcrossing, and genotyping an extensive portfolio of germplasm would not report whether plants were fertile or sterile. Applicant has put together a particular haplotype of genetic and cytoplasmic markers, but has not validated the resulting effect on flowering.
This incomplete description of the claimed invention is notable in Example 1, where Applicant uses active past-tense to partially describe the claimed multi-step method (i.e. describing steps they tested), but then switches to future-tense (i.e. as yet to be performed) in describing the final steps of validating their work.
Looking to the data tables to clarify this unusual wording, which mixes working results/data and prophetic steps within examples, Applicant’s tables only indicate marker haplotypes and/or numbers of haploid or doubled haploid plants recovered. Applicant does not indicate what percentage of recovered plants were functionally male sterile due to the claimed CMS transfer method. No data is presented indicating Applicant has validated the effect of the particular marker haplotype(s) in conferring CMS, as part of the complete method recited in claim 1.
Because of this, the subject matter has not been described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor had possession of the claimed invention (i.e. predictably conferring CMS phenotype).
Claim 15 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
Claim 15 recites the limitation of an anthocyanin marker selected from R1-navajo and R1-SCM2. The state of the art indicates that R1-navajo is a well-known and widely used phenotypic marker in maize doubled-haploid research, but the R1-SCM2 marker is uncommon. One seeking to replicate the claimed method, and particularly the use of anthocyanin markers of claim 15 to identify haploid plants, would require germplasm carrying the recited R1-SCM2 in reconstructing or replicating Applicant’s CHIP line.
Turning to the specification for more information regarding the source and use of the R1-SCM2 marker, Applicant defines “R1-SCM2” as: the R1-SCM2 anthocyanin marker [p.23, l.15]. No reference or citation is specifically made regarding publicly available sources of the marker.
Applicant’s Example 2 describes creation of a ‘CHIP’ with R1-SCM2, wherein they state source material for this marker includes embryos from the population ‘SYN-INBC34 x SYN-INBC34RS’ [p.30, l.43], progeny of ‘SYN-INB77M-CMS’ [p.32, l.1], and ‘RWKS’ [p.32, Table 3]. Applicant then proceeds to describe selection of the marker in various progeny, none of which are identified by line designation or genotype.
Two of these potential sources of R1-SCM2 appear to be working/temporary breeding populations (i.e. ‘SYN-xxx’ populations), with the only fixed breeding line having R1-SCM2 being ‘RWKS’. Search of the disclosure for ‘RWKS’ shows it is also used for transfer of the R1-nj color marker [p.28, l.9-21], but there is no indication of what this line is. It is unclear if ‘RWKS’ is a proprietary line and whether one reading the disclosure would be able to obtain it, and thus make use of the recited R1-SCM2 marker. Applicant provides no direction with respect to sourcing such material.
Turning to the research literature, there are reports of the marker R1-scm2 used as a visual anthocyanin marker, and molecular descriptions of its sequence for genetic studies directed to transposable elements and paramutation (i.e. not in cultivar development) [see p.984, col.1, ¶.4 & Figure 4; p.989, col.1, ¶.1 in Panavas Genetics, Volume 153, Issue 2, 1 October 1999, Pages 979–991]. It is obvious it could be, and has been, used as a phenotypic marker. The literature makes it clear the gene can be used to track parentage phenotypically via anthocyanin patterns, however, there appears to be indication of adapted breeding lines or readily available sources of it.
There also appears to be no prior art referencing the use of ‘RWKS’, or derivatives, carrying this R1-SCM2 marker. There appear to be no working examples of the use of ‘RWKS’ in other breeding reports prior to Applicant’s filing. The line ‘RWKS’ carrying R1-SCM2 is not on sale commercially, and there is no reference indicating public availability of this critical material in a seedbank or accessible repository.
The closest reference is a more recent 2024 publication from Applicant, describing ‘RWKS’ as being derived from the foundational inducer ‘ZMS’ through the intermediate ‘RWS’ [see p.315, col.1, ¶.3 in Delzer et al. The Crop Journal 12(2024) 314-319; Published 21 Dec 2023]. In that report, there is no description of the specific breeding steps or selection process to derive a line similar to ‘RWKS’, what doing so ‘through an intermediate’ indicates with respect to crossing or pedigree structures, or if the ‘RWS’ intermediate itself is obtainable. It remains unclear if ‘RWKS’ carrying R1-SCM2 is a proprietary line held by Applicant.
A plant breeder of ordinary skill reading Applicant’s disclosure and seeking to replicate the described method would first ask themselves where to obtain breeding lines carrying the essential components of the described system, to be combined into the key functional ‘CHIP’ line. This synthesis would include source germplasm carrying (a) CMS, (b) restorer alleles, (c) cenh3 mutations, (d) the R1-Navajo marker, and (e) the R1-SCM2 marker. Multiple sources of CMS, restorer alleles and R1-Navajo in maize breeding lines are publicly available. Generation of cenh3 mutations (i.e. engineered haploid induction) is described by Applicant, and by other research groups. However, there is no clear answer how one would obtain the necessary starting material comprising component (e), the R1-SCM2 marker, which appears to come from ‘RWKS’. Further, there is no answer if one could even recreate a line equivalent to ‘RWKS’ to use as a donor of R1-SCM2 given no clear record of its breeding history.
Applicant has not fully described the critical source material for this component of their proposed inventive method, or presented a reasonable way to predictably obtain it. Applicant has not described a means of obtaining a functionally relevant breeding line comprising R1-SCM2 that could be used to re-create their claimed ‘CHIP’ plant, absent undue experimentation amounting to re-creation of a marker line equivalent to ‘RWKS’. Breeding such a line would require a multi-step, multi-cycle breeding process of which there is no guidance regarding pedigree structures, selection factors, or even access to required foundational germplasm (i.e. ‘RWS’). This represents a burden of undue experimentation and because of this, claim 15 is not enabled and is rejected.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1, 4, 6-7, 31, 33 & 38 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Ren [Plant Biotechnol J, 15: 1361-1370 (2017); Published 10 Aug 2017].
Claim 1 recites conferring CMS by crossing a haploid-inducer line with CMS to a second plant and generating a progeny with CMS cytoplasm and the nuclear genome of the second plant.
Claim 4 adds the limitation that the haploid inducer line is female fertile. The claim recites that the CHIP (i.e. CMS, haploid-inducing line) can be either male sterile or fertile. This relates to the genetic status of the CHIP, and whether it carries a corresponding restorer alleles (Rfs), for the purposes of maintaining a population of viable CMS material (i.e. absent use of Rfs and some portion of male fertile plants, the CMS line would go extinct). As such, the critical limitation with respect to the method of claim 1 is that the CMS line, whether male fertile or not, is able to be used as a maternal parent in a cross and thus transfer its CMS cytoplasm.
Claim 6 adds the limitation that the haploid inducer line is used as a paternal haploid inducer. This requires that the CMS line (CHIP) is used as the maternal parent, and when pollinated by a non-CMS line (DIP), induces the pollen from the non-CMS line to form a haploid plant (i.e. paternal haploid induction).
Claim 7 includes that the line comprises a cenh3 mutation.
Ren discloses this method. They describe use of directed cenh3 mutations to generate haploid inducer lines [p.1365, col.1, ¶.1-2], the combination of those lines with CMS backgrounds for seed production (i.e. Applicant’s ‘CHIP’) is described [p.1366, col.2, ¶.3—p.1367, col.1, ¶.1]. Crossing the CMS haploid induction lines to other inbred lines (i.e. Applicant’s ‘DIPs’) to generate paternal haploids from the inbred (i.e. inducer line is used as maternal parent) is also described [id]. They describe the benefits of this system, and its use in maize [p.1367, col.1, ¶.1, l.10-21].
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 2, 20 & 22 are rejected under 35 U.S.C. 103 as being unpatentable over Ren [Plant Biotechnol J, 15: 1361-1370 (2017); Published 10 Aug 2017] in view of Weider [Crop Sci. 49:77–84 (2009); Published January 2009].
Ren teaches use of directed cenh3 mutations to generate inducer lines [p.1365, col.1, ¶.1-2], the combination of those lines with CMS backgrounds for seed production (i.e. Applicant’s ‘CHIP’) [p.1366, col.2, ¶.3—p.1367, col.1, ¶.1], and crossing the CMS haploid induction lines to other inbred lines (i.e. Applicant’s ‘DIPs’) to generate paternal haploids from the inbred (i.e. inducer line is used as maternal parent) [id]. They describe the benefits of this system, and its use in maize [p.1367, col.1, ¶.1, l.10-21].
Ren does not teach the use of CMS-C, CMS-S, or CMS-T cytoplasm of maize, or the corresponding restorer of fertility genes (Rf3, Rf4, Rf10, Rf11 and Rf12).
Weider teaches the use of CMS-C, CMS-S, & CMS-T cytoplasm in maize and corresponding restorer alleles, including the CMS-C/Rf4 system [p.77, col.2, ¶.3—p.78, col.2, ¶.1]. Weider teaches that many breeders focus on the use of C-cytoplasm in maize because it has distinct qualities relative to CMS-T and CMS-S systems [p.82, col.1, ¶.2—col.2, ¶.1].
Before the effective filing date of the claimed invention, it would have been obvious to one of ordinary skill in the art to modify the method of creating haploid-inducing CMS lines taught by Ren, which can be achieved using mutation of cenh3, to one utilizing maize CMS cytotypes (i.e. CMS-C, CMS-T, CMS-S) and corresponding restorer alleles known to be used in seed production taught by Weider. This would be obvious because Ren teaches one should use a CMS system, and the maize-specific systems of CMS-C, CMS-T & CMS-S are well-known and have established their value and applicability for breeding purposes.
One of ordinary skill in the art would have been motivated to combine these teachings to obtain the described benefit of a system of rapidly deploying CMS in different genetic backgrounds (i.e. DIPs). Doubled-haploid approaches to ‘swapping’ cytoplasm would eliminate the lengthier, costly procedure of traditional backcrossing or conversion to CMS for improved seed production efficiency, as described by Ren [p.1361, col.1, ¶.2—col.2, ¶.1; p.1367, col.1, ¶.1, l.10-21]. Use of maize CMS systems based on three widely-described cytotypes, and specifically CMS-C, was known to have value to corn seed producers and is described by Weider.
Regarding claim 2; Ren teaches use of directed cenh3 mutations to generate inducer lines with CMS cytoplasm, as described in the 102 rejection above [see p.10, ¶.1-3]. Weider teaches use of the CMS-C system directed to maize [p.77, col.2, ¶.3—p.78, col.2, ¶.1].
Regarding claim 20; Weider teaches use of the CMS-C system directed to maize, including the respective Rf4 restorer alleles, as in Applicant’s claims 2, 20 & 22 [p.77, col.2, ¶.3—p.78, col.2, ¶.1]. These male-fertility factors relate to maintenance of the CMS line per se, and therefore any method use of a CMS line would entail maintenance of a viable population of CMS germplasm would require at least a plant comprising a restorer allele of some sort (i.e. absent this, the CMS line would not be able to maintained without outcrossing).
Regarding claim 22; Weider teaches use of Rf4 restorer alleles, and this encompasses the use of null variant of any of these (i.e. allelic forms which render the CHIP male sterile) as in Applicant’s claim 22 [p.77, col.2, ¶.3—p.78, col.2, ¶.1].
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Ren [Plant Biotechnol J, 15: 1361-1370 (2017); Published 10 Aug 2017] in view of Chaikam [Theor Appl Genet (2015) 128:159–171; Published 11 Nov 2014], and Panavas [Genetics, Volume 153, Issue 2, 1 October 1999, Pages 979–991; Published 1 Oct 1999].
Ren teaches use of directed cenh3 mutations to generate inducer lines [p.1365, col.1, ¶.1-2], the combination of those lines with CMS backgrounds for seed production (i.e. Applicant’s ‘CHIP’) [p.1366, col.2, ¶.3—p.1367, col.1, ¶.1], as described above.
Ren does not teach use of maize anthocyanin markers R1-Navajo or R1-SCM2.
Chaikam teaches the use of the R-Navajo anthocyanin marker in production of doubled haploid maize, as well as potential shortcomings to its detection [p.160, col.1, ¶.2—col.2, ¶.1]. Chaikam teaches use with additional phenotypic markers to overcome instances where there is not strong expression of the R-Navajo phenotype [p.162, col.1, ¶.2; col.2, ¶.3—p.163, col.1, ¶.1].
Panavas teaches the use of the R-SCM2 anthocyanin marker in maize to monitor parentage and identification of crossing with particular genetic stocks carrying the marker [p.984, col.1, ¶.4 & Figure 4; p.989, col.1, ¶.1]. They teach R-scm derivatives (i.e. R1-SCM2) can condition fully colored embryos and visible pigmentation of seedling leaves [id], and thus, the use of the R1-SCM2 anthocyanin marker to identify successful crosses/progeny.
Before the effective filing date of the claimed invention, it would have been obvious to one of ordinary skill in the art to modify the method of creating haploid-inducing CMS lines taught by Ren, which can be achieved using mutation of cenh3, to one with known anthocyanin markers used in tracking parentage of maize plants and identifying haploids, taught by Chaikam (i.e. R-Navajo) and Panavas (i.e. R-SCRM2). This would be obvious because Ren teaches a system requiring tracking of the genomic constitution of progeny (i.e. detection of haploids and/or parentage), and the anthocyanin marker systems described by Chaikam and by Panavas had established their utility in as visible phenotypic markers to determine parentage.
One of ordinary skill in the art would have been motivated to combine these teachings to obtain the described benefit of a system of rapidly deploying CMS in different genetic backgrounds (i.e. DIPs). Doubled-haploid approaches to ‘swapping’ cytoplasm would eliminate the lengthier, costly procedure of traditional backcrossing or conversion to CMS for improved seed production efficiency, as described by Ren [p.1361, col.1, ¶.2—col.2, ¶.1; p.1367, col.1, ¶.1, l.10-21]. One would be motivated to apply the known anthocyanin markers that were in use at the time of filing, to Ren’s method directed to the generation of haploid plants (i.e. via cenh3 inducer lines) because such markers are necessary to successfully identify progeny for selection.
Regarding claim 15; The specific marker limitations recited are met by description of anthocyanin-producing alleles of R1-Navajo by Chaikam and R1-SCM by Panavas. Their previous research reports demonstrate their use and successful application as phenotypic markers indicating parentage and/or genetic constitution.
Claims 8, 10 & 12 are rejected under 35 U.S.C. 103 as being unpatentable over Ren [Plant Biotechnol J, 15: 1361-1370 (2017); Published 10 Aug 2017] in view of Lv [Nat Biotechnol 38, 1397–1401 (2020); Published 9 Nov 2020] and Dong [Front. Genome Ed. 3:670529; Published 12 May 2021].
Ren teaches use of directed cenh3 mutations to generate inducer lines [p.1365, col.1, ¶.1-2], the combination of those lines with CMS backgrounds for seed production (i.e. Applicant’s ‘CHIP’) [p.1366, col.2, ¶.3—p.1367, col.1, ¶.1], as described above.
Ren does not teach a heterozygous knockout mutation via gene editing. Ren does not teach the specific SEQ ID NO.5 or 6, or the use of CRISPR-Cas12a enzymes.
Lv teaches the generation of haploid inducer lines via modification of cenh3 using gene editing, including knockouts that result in heterozygous / hemizygous plants [p.1397, col.2, ¶.2—p.1399, col.2, ¶.1]. Lv teaches relevant gene sequence and structure of cenh3, as well as gene-edited mutations, in wheat and other orthologs including maize [p.1398, Fig.1(d)]. Lv teaches this potential method in ‘cytoplasm swapping’ and rapid transfer of CMS in crops such as maize [p.1401, col.1, ¶.1]
Dong teaches the use of CRISPR-Cas12a systems for gene editing in maize [p.2, col.1, ¶.3; p.3, col.1, ¶.3; p.6, Table 3; p.7, Table 5]. They teach CRISPR-Cas12a as an attractive alternative system to CRISPR-Cas9 for genome editing [p.7, col.1, ¶.2].
Before the effective filing date of the claimed invention, it would have been obvious to one of ordinary skill in the art to modify the method of creating haploid-inducing CMS lines taught by Ren, which can be achieved using mutation of cenh3, to one utilizing gene-edited knockouts in cenh3 sequence, or orthologs thereof, taught by Lv, along with known use of Cas12a gene editing systems in maize plants, as in Dong. This would be obvious because Ren teaches that one need disrupt cenh3 function and gene editing was demonstrated by Lv as one, very precise, method of doing that. The use of the alternate Cas12a system would be obvious because it was known to effectively edit genes, particularly in maize, which is the focus of the instant application.
One of ordinary skill in the art would have been motivated to combine these teachings to obtain the described benefit of a system of rapidly deploying CMS in different genetic backgrounds (i.e. DIPs). Doubled-haploid approaches to ‘swapping’ cytoplasm would eliminate the lengthier, costly procedure of traditional backcrossing or conversion to CMS for improved seed production efficiency, as described by Ren [p.1361, col.1, ¶.2—col.2, ¶.1; p.1367, col.1, ¶.1, l.10-21]. Gene editing presents a potentially more rapid, and precise, method of doing this. Further, use of a Cas12a-based system, particularly in maize, was known to provide a beneficial alternative to the gene editing process established via Cas9 systems [Dong, p.7, col.1, ¶.2]. One would be motivated to combine known these elements from the previously described gene editing system(s) for the same purposes as taught by Ren in beneficially generating a haploid-inducing parent by knocking out or disrupting cenh3.
Regarding claim 8; Ren teaches use of directed cenh3 mutations to generate inducer lines with CMS cytoplasm, as described in the 102 rejection previously outlined [see above, p.10, ¶.1-3]. Lv teaches use of gene edited knockouts to generate loss of function mutants, as in Applicant’s claim 8 [p.1397, col.2, ¶.3].
Regarding claim 12; Dong teaches use of the alternate CRISPR-Cas system comprising CRISPR-Cas12a enzyme(s) [p.2, col.1, ¶.3; p.3, col.1, ¶.3; p.6, Table 3; p.7, Table 5; p.7, col.1, ¶.2].
Regarding claim 10; this claim is drawn to SEQ ID NO.5 & 6, which are mutated sequences in cenh3 meant to induce a loss-of-function. The particular sequences recited are routine in design, as the sequence and structure of the conserved cenh3 gene, including orthologs, was previously reported and is described in detail by Lv [p.1398, Fig.1(d)]. One skilled in the art would recognize there are several functionally equivalent ways of arriving at a loss-of-function mutant in any given gene of known sequence, and substituting one functionally equivalent mutation for another is merely a design choice, absent support that there is a non-obvious feature to the particularly claimed mutation which renders it distinct from the prior art.
Conclusion
No claims are allowed.
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/KEITH R. WILLIAMS/Examiner, Art Unit 1663
/Amjad Abraham/SPE, Art Unit 1663
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