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 .
Priority
Acknowledgment is made of applicant’s claim for priority to Provisional Application No. 63394019, filed on 08/01/2022.
Status of the Claims
Amendments dated 01/12/2026 are entered.
Claims 5-7, 9-10,12, 14-18, 20, 23, 25, 25, 27, 29, and 32-34 are cancelled.
Claims 1-4, are pending and are examined herein.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 01/31/2025 is acknowledged and is being considered by the examiner.
Drawings
The drawings are objected to because though Figure 1 is pertinent to the method steps, it is not visually discernible so as for one to readily learn the degree or severity of “wounding the exposed, developing embryos” (c.f., claim 4).
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Objections
Claims 1 and 19 are objected to because of the following informalities:
“Agrobacterium” should be italicized.
Claims 2, 22, 24, 26, and 28 are objected to because of the following informalities:
Scientific names of plant genera should be italicized.
Claims 3-4 are objected to because of the following informalities:
Line 1 should recite “claim 1” instead of “claims 1”
Claim 11 is objected to because of the following informalities:
The acronym PSI is used without being defined or spelled out
Claim 30 is objected to because of the following informalities:
Line 1 should recite “claims 1, 19, or 21” instead of “claims 1 19, or 21”
Claims 30-31 are also objected to under 37 CFR 1.75(c) as being in improper form because a multiple dependent claim cannot depend from any other multiple dependent claim. In the instant application claim 30, a multiple dependent claim, depends from claim 21, another multiple dependent claim, and claim 31 depends from claim 30. See MPEP § 608.01(n). In the interest of compact prosecution and as a curtesy to Applicant, claims 30-31 have been examined. However, Applicant is not relieved from the responsibility to respond to this objection and amend the claims
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
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.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
The following is a quotation of 35 U.S.C. 112(b):
Claims 1-4, 8, 11, 13, 19, 21-22,24, 26, 28, and 30-31 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. All dependent claims are included in these rejections unless they contain a limitation that overcomes the deficiencies of the parent claim from which they depend. Details are listed below.
Regarding the rejection of claims 1-4, 8, 11, 13, 19, 21-22,24, 26, 28, and 30-31, the preamble in claim 1 recites a method of transforming a wheat or other cereal in planta. However, the claim further recites active steps “the method comprising: (i) co-incubating, post-anthesis, a cereal inflorescence of developing embryos with an inoculation medium, wherein the inoculation medium comprises a transgene-modified Agrobacterium; (ii) collecting, upon the cereal inflorescence reaching maturity, seeds produced from at least a portion of the developing embryos; and (iii) selecting one or more transgenic seeds from among the collected seeds.” The recited active steps do not accomplish “transforming a wheat or other cereal in planta.” Co-incubating (1) plant tissue with (2) transgene-modified Agrobacterium does not in and of itself necessarily result in a transformed cereal plant or transforming all possible cereal plants. Although the Agrobacterium-mediated transformation is highly efficient in dicotyledons, its application in monocotyledons continues to be problematic. The claim is missing a step of transferring the transgene into the plant tissue, to provide the nexus.
Further, regarding step (iii) of claim 1, how is “selecting one or more transgenic seeds from among the collected seeds’ accomplished if no transgene was in introduced into the cereal inflorescence of developing embryos or the embryos? It is unclear what there is to select in a method of “transforming a wheat or other cereal in planta” where no transgene was introduced into a target plant. Stress signals? hormone levels? Priming against pathogens? Agrobacteria? In all possible cereal plants, in planta?
Claim 1 also recites “seeds produced from at least a portion of the developing embryos” on lines 6-7. It is unclear what the phrase “at least a portion of the developing embryos” means in the context of method of transforming wheat or other cereal in planta comprising the collection of seeds. Is it a numerical subset of embryos in the cereal inflorescence; and if so, how is it determined? A cell on each developing embryo? Some cells? The entire embryo? Some components; DNA, protein? Combinations thereof?
Claim 3, similarly to Claim 1, recites the in the following context: “exposing at least a portion of the developing embryos.” It is unclear what the phrase “at least a portion of the developing embryos” means in this context. What is exposed? Is it a numerical subset of produced embryos in the cereal inflorescence? A cell on each developing embryo? Some cells? The entire embryo? Some components; DNA, protein? Combinations thereof?
Claims 2, 21-22, 24, 26, and 28 are further rejected. Claim 2 recites “[t]he method of claim 1 wherein the cereal is selected from Triticum (wheat), Sorghum (sorghum), Oryza (rice), Avena (oats), Zea (corn), Hordeum (barley), and Secale (rye).” It is unclear whether what is inside the parentheses are intended to be claim limitations. What is outside of the parentheses appears to be genera, which should be italicized. Genera include multiple species and multiple genotypes. Some pairings of what is inside the parentheses and what is outside of the parentheses, genera, share the same scope, while others do not share the same scope (see the list below). The metes and bounds of the claim are unclear. It is suggested that the species of cereal be stated as opposed to genera. Claims 22, 24, 26, and 28 are similarly rejected for recitation of genera followed by a common name for a cereal in parenthesis.
Merriam Webster defines wheat as “any of various Old World annual grasses (genus Triticum, especially T. aestivum and T. turgidum) of wide climatic adaptability that are cultivated in most temperate areas for the wheat they yield.”
Merriam Webster defines sorghum as “any of an economically important genus (Sorghum) of Old World tropical grasses similar to corn in habit but with the spikelets in pairs on a hairy rachis such that sorghum has the same scope compared to Sorghum.
Merriam Webster defines rice as “the starchy seeds of an annual southeast Asian cereal grass (Oryza sativa) that are cooked and used for food” such that rice has a narrower scope compared to Oryza.
Merriam Webster defines oats as “any of several grasses (genus Avena) especially : a widely cultivated cereal grass (A. sativa)” such that oats has the same scope compared to Avena.
Merriam Webster defines corn as “a tall annual cereal grass (Zea mays) originally domesticated in Mexico and widely grown for its large elongated ears of starchy seeds” such that corns has a narrower scope compared to Zea.
Merriam Webster defines barley as “ a cereal grass (genus Hordeum and especially H. vulgare) having the flowers in dense spikes with long awns and three spikelets at each joint of the rachis.” such that barley has a the same scope compared to Hordeum.
Merriam Webster defines rye as “a hardy annual grass (Secale cereale) that is widely grown for grain and as a cover crop” such that rye has a narrower scope compared to Secale.
Claims 4, 8, 11, 13, 21, and 30-31 are further rejected because claims 4, 8, 11, and 13 recite the term “about” in relation to a numeric data point. Paragraph 0088 of the specification recites the term ‘about’ “can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1 % of a stated value or of a stated limit of a range.” This recitation is too ambiguous to redefine "about” particularly because of the “can allow” phrase nested therein. See MPEP 2111.01 subsection IV.
Claim 8, which depends from claim 1, recites a method step wherein the cereal inflorescence is “covered until maturity” after co-incubation with the inoculation medium. It is unclear what covered means in a method of transforming a wheat or other cereal. What is the purpose of the covering? To place the cereal inflorescence in the dark? to lower gaseous exchange? to reduce Carbon dioxide availability? To retain liquids or impede evaporation? Or a combination thereof? The purpose for which the inflorescence is covered informs the material with which covering is done. Opaque? Clear? semipermeable? Permeable? Airtight? Or a combination thereof?
Further, regarding “covered until maturity,” it is unclear what is meant by maturity making it unclear when the coverage ends. There is no definition of maturity in the specification. Is it structural maturity? Developmental maturity? A functional state as in capable of germinating? What components are present at maturity? How long does it take to get to maturity? "Maturity" is a subjective, variable term in embryology, including plant embryology. The metes and bounds of the claim scope are ambiguous.
Claim 21, which depends from claims 1- 4, recites a method step wherein the vector is a combination of pCAMBIA1305.1 and PIP2-GUS-Bar (c.f., line 2 of claim 21: “a vector selected from pCAMBIA1305. l, PIP2-GUS-Bar, or a combination thereof.”). It is unclear what a combination thee of in a method for transforming cereal using a vector means. Are pCAMBIA1305.1 and PIP2-GUS-Bar wholly combined? If not, what is the ratio of the combination between the two? Which sequences from each vector? Or are the vectors used in tandem, combined use?
Claims 22, 24, 26, and 28 are further rejected. Claim 22 recites “[t]he method of claim 2, wherein the cereal is a Triticum (wheat) variety, wherein the variety can be selected from AG0762, Apogee [,etc.]” It is unclear what “the variety can be selected from” is intended to be. A closed grouping? Or are other varieties not listed here that are encompassed by the claim? Thus, the metes and bounds of the claim are unclear. Claims 24, 26, and 28 are similarly rejected for the recitation of “the variety can be selected from.” The phrases in claims 22, 24, 26, and 28 beginning from “wherein the variety can be selected from” are not given patentable weight because it is unclear what conditions are necessary for these varieties to be selected.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claim 31 is rejected under 35 U.S.C. 101 because the claimed invention is directed to a product of nature without significantly more. The claim(s) recite(s) "a cell, a tissue, an organ, or a seed (or T2 plant obtained therefrom) obtained from the transgenic plant". This judicial exception is not integrated into a practical application because the claims do not recite any further limitations on the "cell,…tissue, … organ, or … seed (or T2 plant obtained therefrom) obtained from the transgenic plant". The claim(s) does/do not include additional elements that are sufficient to amount to significantly more than the judicial exception because the claims do not recite any further limitations on the "cell,…tissue, … organ, or … seed (or T2 plant obtained therefrom) obtained from the transgenic plant."
Due to sexual hybridization and Mendelian segregation, some of the progeny (i.e., “seed …or T2 plant obtained therefrom”) will be indistinguishable from a product of nature. In the case of chimeric transformations, some tissue from transgenic plants where some tissues are transgenic while others remain wild-type (untransformed) from seed or tissue transformation.
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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-4, 8, 11, 22, 24, and 28 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by KWS (U.S. Patent Application Publication No. 2018/0142248 A1, assigned to KWS SAAT SE and Co KGaA, titled 'Methods for the in planta transformation of plants and manufacturing processes and products based and obtainable thereform' [sic], published 2018).
Regarding claim 1, KWS discloses “in planta transformation methods for plants or plant materials with a genetic construct" “ wherein the plant…is…from a species selected from the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum officinarium, Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum,…” (i.e., a method of transforming a wheat or other cereal in planta) (Abstract; claims 16 and 25).
KWS also teaches that the “methods as disclosed [therein] can be adapted to other flowers or inflorescences of other crops based on the disclosure provided [therein]” (paragraph 0078) where the “localization of a wheat immature inflorescence in two different wheat cultivars” (paragraph 0040; FIG. 9) is also taught.
Regarding step (i) of claim 1, KWS teaches that “Flower spikes [inflorescences] were obtained from bolted plants grown in the greenhouse 14 days after anthesis” (i.e., post-anthesis) (paragraph 0176) and that “for the purpose of the present invention,…Agrobacteria a carrying a virus encoding T - DNA have been introduced into plant target structure of interest via filtration” (0161) .
Further regarding step (i) of claim 1, in paragraph 0172, KWS teaches that "the susceptibility of immature tassel tissues for Agrobacterium (Ab) infection was tested [using a] construct expressing a red fluorescent…to transform an immature tassel [where the] bacteria were grown to an OD600 of 1.0 and the tassel was inoculated for 10 minutes" (i.e., co-incubating a cereal inflorescence of developing embryos with an inoculation medium, wherein the inoculation medium comprises a transgene-modified Agrobacterium)(also see paragraph 0159). “The plants were on a V6-V7 stage and the tassels were between 2 and 3 cm long” (paragraph 0172).
Regarding step ii of claim 1, KWS teaches that “seeds from tassel bombarded plants were produced and the result of the targeted modification was analyzed in T1 plants" (paragraph 0157), and that "mature seed pods were collected after 3-4 weeks" (paragraph 0222) (i.e., collecting, upon the cereal inflorescence reaching maturity, seeds produced from at least a portion of the developing embryos).
Regarding step iii of claim 1, KWS teaches that " seeds from tassel bombarded plants were produced and the result of the targeted modification was analyzed in T1 plants [and that] it was thus possible to detect a positive event in 51 T1 plants by analyzing the seeds from one mother plant" (i.e., selecting one or more transgenic seeds from among the collected seeds) (paragraph 0157).
wherein the inoculation medium comprises a transgene-modified Agrobacterium (para [0176] - "Flower spikes were obtained from bolted plants grown in the greenhouse 14 days after anthesis"; para [0172] - "the susceptibility of immature tassel tissues for Agrobacterium (Ab) infection was tested. A construct expressing a red fluorescent was used to transform an immature tassel cut and separated from the plant. The plants were on a V6-V7 stage and the tassels were between 2 and 3 cm long. The bacteria were grown to an OD600 of 1.0 and the tassel was inoculated for 10 minutes"; para [0047]- "FIG. 16 A and B (FIG. 16 A and 8) shows the gel analysis of a PCR for verifying the successful integration of DNA inserted into tassel meristematic cells of interest for two different transgenes");
Regarding claim 2, KWS teaches claim 25 which recites “wherein the plant or the plant material is or is part of a plant from a species selected from the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor,…Zea mays,…Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticale, Secale cereale,…Hordeum marinum” and claim 27 which recites “the immature inflorescence is a male inflorescence (tassel) of a Zea mays plant, or wherein the immature infloresecene is the inflorescence of a Triticum aestivum plant” (i.e., wherein the cereal is selected from Triticum (wheat), Sorghum (sorghum), Oryza (rice), Zea (corn), Hordeum (barley), and Secale (rye)). See claim 25 of KWS. Also see MPEP 2131.02.
Regarding claim 3, KWS teaches that “said in planta transformation can be conducted with a plant, plant cell, plant tissue or plant material that was specifically prepared or exposed for the transformation process still being in connection with the living plant” (i.e., exposing at least a portion of the developing embryos prior to co-incubation)(paragraph 0048).
KWS also teaches:
"exposing the at least one meristematic cell of the meristematic tissue of the immature inflorescence"(paragraph 0077);
FIG. 1, which “shows a schematic diagram of the maize immature inflorescence, the tassel and illustrates details of a spikelet pair [where] each spikelet contains two florets, the upper floret and the lower floret enclosed by two glumes) (paragraph 0032);
"the [KWS] invention specifically targeting at least one meristematic cell of a plant inflorescence as long as meristematic cells are present. For certain approaches, it might be favorable to do the transformation in an earlier developmental state of the inflorescence development to (i) potentially affect a larger cell population of the progenies of the at least one meristematic cell to be transformed and/or (ii) depending on the type of transformation chosen to have less cell layers (glumes, lemma or palea or the epidermis of the anther) surrounding the target tissue or cell" (paragraph 0130);
that “as the meristem is completely covered by leaves in seedlings, it has to be dissected first to be exposed for the further treatment. To this end, the surrounding tissue structures have to be removed to expose the meristem (indicated with an arrow in FIG. 2)" (paragraph 0033).
These are comparable to lines 2-3 of claim 3 which recite “exposing comprises trimming awns, removing outer glumes, and plucking out middle florets of each respective embryo.” KWS does not explicitly teach the trimming of awns nor the plucking of middle florets. However, KWS does teach the removal of surrounding structures in order to expose the target structure (see bullet list above). Additionally, FIGs 12 and 15 of KWS show immature maize embryos and extracted immature wheat embryos without awns, outer glumes, and middle florets (i.e., exposing comprises trimming awns, removing outer glumes, and plucking out middle florets of each respective embryo).
Regarding claim 4, KWS teaches that “Immature kernels are collected from immature ears 5 to 20 days after anthesis [(i.e., about 7 days to about 24 days after anthesis), the] kernels are surface sterilized using bleach and ethanol, [and then] The immature embryos are then extracted with a scalpel under a microscope [(i.e., wounding)]. Those meristems are then targeted by the different transformation methodologies[(i.e., prior to co-incubation)]” (paragraph 0182).
KWS also teaches "[making] some cuts in the meristem area in order to facilitate the access of different transformation methods (e.g. microinjection, Agrobacterium) ... Those exposed meristems can be treated with substances that can diminish the physical damage and increase survival, like antioxidants. Once the method of transformation has been applied[(i.e., prior to co-incubation)], the wound can be closed by physical methods (covering with a tissue, parafilm or other materials)" (paragraph 0179).
Regarding claim 8, which depends from claim 4, KWS teaches that “in order to avoid Agrobacterium overgrowth, 2 to 7 days after injection a solution of antibiotics (e.g. timentin, carbenicillin, cefotaxime, etc.) was applied to the infected tissue” (i.e., the cereal inflorescence is co-incubated with the inoculation medium for about 10 hours to about 72 hours) (paragraph 0172).
KWS also teaches that “[after] the delivery method [a method of transformation] was…applied to the exposed meristematic tissues[,] treated zones were covered and let to grow (paragraph 0201). In “Example 6: Optimization of Further Parameters” paragraph 0165 KWS teaches that “Depending on the plant species chosen for transformation and depending on transformation methods, several parameters were adjusted to achieve an improved transformation efficiency,” adding in paragraph 0167 that “it is desirable to remove the Agrobacteria by known methods a few days after transformation.” In paragraph 0176< KWS teaches that “once the meristem is hit, a time for recovery and embryo maturation has to be done. This embryo maturation takes place in an incubator in the dark [(i.e., and then covered until maturity).”
Regarding claim 11, after disclosing that “the tassel tissue or cells thereof can be transformed with… Agrobacterium mediated transformation,” KWS teaches that “the A . tumefaciens… inoculated seeds were then placed on filter papers on wet perlite in flasks covered with aluminum foil and incubated at 23° C,” and that “each treated inflorescence was covered with a plastic bag ( to maintain humidity ) at 25° C” (i.e., co-incubated with the inoculation medium at a temperature of about 25 ± 5 °C) (paragraphs 0159 and 0202).
Regarding claim 22, which depends from claim 2, KWS teaches in claim 26 that “the plant or the plant material is or is part of a plant from any variety or subspecies belonging to a species selected from the group consisting of… Triticum aestivum” (i.e., wherein the cereal is a Triticum (wheat) variety). Refer to the 112b rejection of claims 22, 24, 26, and 28 in this Office Action.
Regarding claim 24, which depends from claim 2, KWS teaches in claim 26 that “the plant or the plant material is or is part of a plant from any variety or subspecies belonging to a species selected from the group consisting of Hordeum vulgare, Hordeum bulbusom,…”(i.e., wherein the cereal is a Hordeum (barley) variety). Refer to the 112b rejection of claims 22, 24, 26, and 28 in this Office Action.
Regarding claim 28, which depends from claim 2, KWS teaches in claim 26 that “the plant or the plant material is or is part of a plant from any variety or subspecies belonging to a species selected from the group consisting of …Sorghum bicolor,…” (i.e., wherein the cereal plant is a Sorghum (sorghum) variety). Refer to the 112b rejection of claims 22, 24, 26, and 28 in this Office Action.
Accordingly, KWS anticipates the claimed inventions.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1-4, 8, 11, 13, 19, 21-22,24, 26, 28, and 30-31 rejected under 35 U.S.C. 103 as being unpatentable over Shen (U.S. Patent Application Publication No. 2012/0322122 A1, assigned to Hui Shen, titled ' Compositions and methods for improved plant feedstock', published 2012) in view of KWS (U.S. Patent Application Publication No. 2018/0142248 A1, assigned to KWS SAAT SE and Co KGaA, titled 'Methods for the in planta transformation of plants and manufacturing processes and products based and obtainable thereform', published 2018) taken with the evidence of Jones (Jones HD, Doherty A, Wu H. Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat. Plant Methods. 2005 Sep 5;1(1):5. doi: 10.1186/1746-4811-1-5. PMID: 16270934; PMCID: PMC1277018., published 2005).
Regarding claim 1, Shen teaches that “Agrobacterium-mediated transformation techniques have now been applied to rice…, wheat…, barley…, alfalfa …, and maize …” (i.e., a method of transforming a wheat or other cereal) (paragraph 0080).
Shen also teaches that “Embryonic callus generated from immature inflorescences of switchgrass cv Alamo line ST2 was transformed by Agrobacterium-mediated transformation” (paragraph 0141).
Shen does not explicitly teach a method of transforming a wheat or other cereal in planta. Nor does Shen explicitly teach the method comprising: (i) co-incubating, post-anthesis, a cereal inflorescence of developing embryos with an inoculation medium, wherein the inoculation medium comprises a transgene-modified Agrobacterium; (ii) collecting, upon the cereal inflorescence reaching maturity, seeds produced from at least a portion of the developing embryos; and (iii) selecting one or more transgenic seeds from among the collected seeds.
However, KWS is also in the field of plant transformation and teaches “in planta transformation methods for plants or plant materials with a genetic construct" “ wherein the plant…is…from a species selected from the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum officinarium, Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum,…” (i.e., a method of transforming a wheat or other cereal in planta) (Abstract; claims 16 and 25).
In paragraph 0014 KWS recites “an outstanding need to create stable or transient transformation of a plant cell or a plant material in planta which directly yields transformed meristematic structures in a plant within an inflorescence meristematic cell or tissue, as this would pave the way for the direct acquisition of plant reproductive material directly in the modified plant and not only from the progeny thereof or after cumbersome and expensive in vitro cultivation.”
KWS also teaches that the “methods as disclosed [therein] can be adapted to other flowers or inflorescences of other crops based on the disclosure provided [therein]” (paragraph 0078) where the “localization of a wheat immature inflorescence in two different wheat cultivars” (paragraph 0040; FIG. 9) is also taught.
Regarding step (i) of claim 1, KWS teaches that “Flower spikes [inflorescences] were obtained from bolted plants grown in the greenhouse 14 days after anthesis” (i.e., post-anthesis) (paragraph 0176) and that “for the purpose of the present invention,…Agrobacteria a carrying a virus encoding T - DNA have been introduced into plant target structure of interest via filtration” (0161) .
Further regarding step (i) of claim 1, in paragraph 0172, KWS teaches that "the susceptibility of immature tassel tissues for Agrobacterium (Ab) infection was tested [using a] construct expressing a red fluorescent…to transform an immature tassel [where the] bacteria were grown to an OD600 of 1.0 and the tassel was inoculated for 10 minutes" (i.e., co-incubating a cereal inflorescence of developing embryos with an inoculation medium, wherein the inoculation medium comprises a transgene-modified Agrobacterium)(also see paragraph 0159). “The plants were on a V6-V7 stage and the tassels were between 2 and 3 cm long” (paragraph 0172).
Regarding step ii of claim 1, KWS teaches that “seeds from tassel bombarded plants were produced and the result of the targeted modification was analyzed in T1 plants" (paragraph 0157), and that "mature seed pods were collected after 3-4 weeks" (paragraph 0222) (i.e., collecting, upon the cereal inflorescence reaching maturity, seeds produced from at least a portion of the developing embryos).
Regarding step iii of claim 1, KWS teaches that " seeds from tassel bombarded plants were produced and the result of the targeted modification was analyzed in T1 plants [and that] it was thus possible to detect a positive event in 51 T1 plants by analyzing the seeds from one mother plant" (i.e., selecting one or more transgenic seeds from among the collected seeds) (paragraph 0157).
wherein the inoculation medium comprises a transgene-modified Agrobacterium (para [0176] - "Flower spikes were obtained from bolted plants grown in the greenhouse 14 days after anthesis"; para [0172] - "the susceptibility of immature tassel tissues for Agrobacterium (Ab) infection was tested. A construct expressing a red fluorescent was used to transform an immature tassel cut and separated from the plant. The plants were on a V6-V7 stage and the tassels were between 2 and 3 cm long. The bacteria were grown to an OD600 of 1.0 and the tassel was inoculated for 10 minutes"; para [0047]- "FIG. 16 A and B (FIG. 16 A and 8) shows the gel analysis of a PCR for verifying the successful integration of DNA inserted into tassel meristematic cells of interest for two different transgenes");
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine and modify the transformation teachings of Shen with the in planta transformation teachings of KWS, arriving at the claimed invention. One would have been motivated to do in order to “[acquire] plant reproductive material directly in the modified plant and not only from the progeny thereof or after cumbersome and expensive in vitro cultivation.
Regarding claim 2, which depends from claim 1, KWS teaches claim 25 which recites “wherein the plant or the plant material is or is part of a plant from a species selected from the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor,…Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticale, Secale cereale, Malus domestica, Brachypodium distachyon, Hordeum marinum” and claim 27 which recites “the immature inflorescence is a male inflorescence (tassel) of a Zea mays plant, or wherein the immature infloresecene is the inflorescence of a Triticum aestivum plant” (i.e., wherein the cereal is selected from Triticum (wheat), Sorghum (sorghum), Oryza (rice), Zea (corn), Hordeum (barley), and Secale (rye)). See claim 25 of KWS. Also see MPEP 2131.02.
Regarding claim 3, which depends from claim 1, KWS teaches that “said in planta transformation can be conducted with a plant, plant cell, plant tissue or plant material that was specifically prepared or exposed for the transformation process still being in connection with the living plant” (i.e., exposing at least a portion of the developing embryos prior to co-incubation)(paragraph 0048).
KWS also teaches:
"exposing the at least one meristematic cell of the meristematic tissue of the immature inflorescence"(paragraph 0077);
FIG. 1, which “shows a schematic diagram of the maize immature inflorescence, the tassel and illustrates details of a spikelet pair [where] each spikelet contains two florets, the upper floret and the lower floret enclosed by two glumes) (paragraph 0032);
"the [KWS] invention specifically targeting at least one meristematic cell of a plant inflorescence as long as meristematic cells are present. For certain approaches, it might be favorable to do the transformation in an earlier developmental state of the inflorescence development to (i) potentially affect a larger cell population of the progenies of the at least one meristematic cell to be transformed and/or (ii) depending on the type of transformation chosen to have less cell layers (glumes, lemma or palea or the epidermis of the anther) surrounding the target tissue or cell" (paragraph 0130);
that “as the meristem is completely covered by leaves in seedlings, it has to be dissected first to be exposed for the further treatment. To this end, the surrounding tissue structures have to be removed to expose the meristem (indicated with an arrow in FIG. 2)" (paragraph 0033).
These are comparable to lines 2-3 of claim 3 which recite “exposing comprises trimming awns, removing outer glumes, and plucking out middle florets of each respective embryo.” KWS does not explicitly teach the trimming of awns nor the plucking of middle florets. However, KWS does teach the removal of surrounding structures in order to expose the target structure (see bullet list above). Additionally, FIGs 12 and 15 of KWS show immature maize embryos and extracted immature wheat embryos without awns, outer glumes, and middle florets (i.e., exposing comprises trimming awns, removing outer glumes, and plucking out middle florets of each respective embryo).
Regarding claim 4, which depends from claim 1, KWS teaches that “Immature kernels are collected from immature ears 5 to 20 days after anthesis [(i.e., about 7 days to about 24 days after anthesis), the] kernels are surface sterilized using bleach and ethanol, [and then] The immature embryos are then extracted with a scalpel under a microscope [(i.e., wounding)]. Those meristems are then targeted by the different transformation methodologies[(i.e., prior to co-incubation)]” (paragraph 0182).
KWS teaches that “flower spikes were obtained from bolted plants grown in the greenhouse 14 days after anthesis [(i.e., about 7 days to about 24 days after anthesis), and] immature embryos (lEs) [(i.e., developing embryos)] were isolated from flower stalks [such that] in those immature embryos the shoot apical meristem can be targeted directly (like with microinjection[i.e., wounding]) using a microscope to detect the meristem area or with more random targeting technologies like bombardment [(i.e., wounding), and] once the meristem is hit, a time for recovery [(i.e., prior to co-incubation)] and embryo maturation has to be done” (paragraph 0176).
KWS also teaches "[making] some cuts in the meristem area in order to facilitate the access of different transformation methods (e.g. microinjection, Agrobacterium) ... Those exposed meristems can be treated with substances that can diminish the physical damage and increase survival, like antioxidants. Once the method of transformation has been applied[(i.e., prior to co-incubation)], the wound can be closed by physical methods (covering with a tissue, parafilm or other materials)" (paragraph 0179).
Regarding claim 8, which depends from claim 4, KWS teaches that “in order to avoid Agrobacterium overgrowth, 2 to 7 days after injection a solution of antibiotics (e.g. timentin, carbenicillin, cefotaxime, etc.) was applied to the infected tissue” (i.e., the cereal inflorescence is co-incubated with the inoculation medium for about 10 hours to about 72 hours) (paragraph 0172). and that .
KWS also teaches that “[after] the delivery method [a method of transformation] was…applied to the exposed meristematic tissues[,] treated zones were covered and let to grow (paragraph 0201). In “Example 6: Optimization of Further Parameters” paragraph 0165 KWS teaches that “Depending on the plant species chosen for transformation and depending on transformation methods, several parameters were adjusted to achieve an improved transformation efficiency,” adding in paragraph 0167 that “it is desirable to remove the Agrobacteria by known methods a few days after transformation.” In paragraph 0176 KWS teaches that “once the meristem is hit, a time for recovery and embryo maturation has to be done. This embryo maturation takes place in an incubator in the dark [(i.e., and then covered until maturity).”
Regarding claim 11, after asserting that “the tassel tissue or cells thereof can be transformed with Agrobacterium, including Agrobacterium tumefaciens or Agrobacterium rhizogenes mediated transformation to Agrobacterium mediated transformation,” KWS teaches that “this kind of transformation is well known to the person having skill in the art (see e.g. Jones, H. D. et al., “Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat”, plant methods, 2005…)” (paragraph 0159).
Jones teaches table 1, “summary of main parameters reported for Agrobacterium-mediated transformation of wheat” which includes column headers “Agrobacterium strain (binary vector),” “Inoculation (Co-culture) *rt – room temp,” and “Control of Agrobacterium cells” where multiple rows list Co-culture (i.e., co-incubation) durations and temperature ranges that overlap with “about 10 hours to about 72 hours” of claim 8 and “about 25 ± 5 °C” of claim 11 (page 2). Note that claim 27 recite “wherein the immature inflorescence is a male inflorescence (tassel) of a Zea mays plant, or wherein the immature inflorescence is the inflorescence of a Triticum aestivum plant” such that KWS taken with the evidence of Jones is consonant with the following limitation of claim 11: “wherein the cereal inflorescence is co- incubated with the inoculation medium at a temperature of about 25 ± 5 °C.”
Regarding claim 13, which depends from claim 11, Shen teaches in paragraph 0168 that “leaf strips were put into an enzyme solution composed of cellulase and macroenzyme, vacuum infiltrated for 20 minutes” and KWS teaches in paragraph 0161 that “or the purpose of the present invention, virus particles, in vitro transcripts of viruses or Agrobacteria carrying a virus encoding T-DNA have been introduced into a plant target structure [inflorescence] of interest via filtration (vacuum and non-vacuum)” (i.e., cereal inflorescence of is co- incubated with the inoculation medium under vacuum infiltration).
KWS also teaches that “first, the susceptibility of immature tassel tissues or Agrobacterium (Ab) infection was tested [where] the bacteria were grown to an OD600 of 1.0 and the tassel was inoculated for 10 minutes” (i.e., from about 10 minutes to about 60 minutes)(paragraph 0172).
KWS teaches “[an observation] that higher pressures…damage the tassel branches,” adding in a paragraph headed “Example 6: Optimization of Further Parameters” that “depending on the target structure to be transformed the shot pressure can be in the range of about 10 to 1500 psi, preferably in the range of about 10 to 1000 psi, more preferably in the range of about 10 to 500 psi, even more preferably in the range of about 50 to 400 psi and most preferably in the range of about 50 to 300 psi” (i.e., about 55 PSI) (paragraphs 0155 and 0165).
Neither Shen nor KWS explicitly teach that the vacuum infiltration of co-incubating of a cereal inflorescence of developing embryos with an inoculation medium occurs at 55 PSI from about 10 minutes to about 60 minutes. However, the pressure and duration of the vacuum infiltration are empirically determined and is an optimization of process parameters. According to section 2144.05 of the MPEP, “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). See also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 (“The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages.”).
A particular parameter must first be recognized as a result-effective variable, i.e., a variable, which achieves a recognized result, before the determination of the optimum or workable ranges of said variable might be characterized as routine experimentation. In re Antonie, 559 F.2d 618, 195 USPQ 6 (CCPA 1977). Prior arts teach the use of vacuum infiltration to introduce solutions, including but not limited to Agrobacterium or chemical agent, deep into plant tissue by removing air from intercellular spaces via a vacuum, therefore, determining the pressure and duration of the vacuum infiltration with is routine experimentation depending on the plant or plant tissue to be targeted.
Regarding claim 19, KWS teaches Jones and Jones teaches table 1, which lists C58C1, AGL1 and LBA4404 Agrobacterium strains (i.e., the Agrobacterium is a strain selected from the group consisting of AGL1, LBA4404, and C58C1).
Regarding claim 21, which depends from any one of claims 1-4, pCAMBIA1305.1 has been known in the art for more than a decade. See for example, Shen is published in 2012 and teaches “the pCAMBIA1305.1 backbone (www.cambia.org/daisy/cambia/585.html)” (i.e., the transgene is in a vector selected from pCAMBIA1305.1) ((paragraph 0173).
KWS, additionally, teaches that “a variety of plasmid vectors for use in different target cells of interest is commercially available and the modification thereof is known to the skilled person in the respective field” (paragraph 0053).
Regarding claim 22, which depends from claim 2, KWS teaches in claim 26 that “the plant or the plant material is or is part of a plant from any variety or subspecies belonging to a species selected from the group consisting of… Triticum aestivum” (i.e., wherein the cereal is a Triticum (wheat) variety). Refer to the 112b rejection of claims 22, 24, 26, and 28 In this Office Action.
Regarding claim 24, which depends from claim 2, KWS teaches in claim 26 that “the plant or the plant material is or is part of a plant from any variety or subspecies belonging to a species selected from the group consisting of Hordeum vulgare, Hordeum bulbusom,…”(i.e., wherein the cereal is a Hordeum (barley) variety). Refer to the 112b rejection of claims 22, 24, 26, and 28 In this Office Action.
Regarding claim 26, which depends from claim 2, Shen teaches in paragraph 0085 that “example species for which have been transformed by microprojectile bombardment include monocot species such as maize…, barley …, wheat…, rice…, oat…, rye….”
KWS teaches “in planta transformation methods for plants or plant materials with a genetic construct" “ wherein the plant…is…from a species selected from the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum officinarium, Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum,…” (Abstract; claims 16 and 25). All of these plants are “economically important plants…in the family of Poacea/Graminaceae” (paragraph 0005).
Niether Shen nor KWS explicitly teach that the cereal plant is an Avena (oats) variety. However, the cereal plant, Avena (oats), is also an economically important plant in the family of Poacea/Graminaceae and transformation methods that worked on other cereals in the Poacea/Graminaceae family such as maize, barley, wheat, rice, and rye, have also been shown to work in oats. Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to apply the transformation method disclosed in Shen modified by KWS to Avena (oats) with a reasonable expectation of success.
Regarding claim 28, which depends from claim 2, KWS teaches in claim 26 that “the plant or the plant material is or is part of a plant from any variety or subspecies belonging to a species selected from the group consisting of …Sorghum bicolor,…” (i.e., wherein the cereal plant is a Sorghum (sorghum) variety). Refer to the 112b rejection of claims 22, 24, 26, and 28 In this Office Action.
Regarding claims 30-31, which ultimately depend from claim 1, Shen teaches the following in paragraphs 0104-0107:
“(a) plant seeds of the first (starting line) and second (donor plant line that comprises a transgene of the invention) parent plants[(i.e., claim 30 step (iv) growing a plant from each of one or more of the transgenic seeds of (iii))]…;
(c) pollinate a flower from the first parent plant with pollen from the second parent plant; and
(d) harvest seeds produced on the parent plant bearing the fertilized flower[(i.e., claim 30 step (v) collecting seeds from the plants of (iv))].”
Shen further teaches the following in paragraphs 0108-0111:
“a) crossing a plant of a first genotype containing a desired gene, DNA sequence or element [(i.e., claim 30 step (iv)’s plant from each of one or more of the transgenic seeds of (iii))] to a plant of a second genotype lacking the desired gene, DNA sequence or element;
(b) selecting one or more progeny plant containing the desired gene, DNA sequence or element;
(c) crossing the progeny plant to a plant of the second genotype; and
(d) repeating steps (b) and (c) [(i.e., claim 30 step (vi) selecting for transgenic seeds among the collected seeds of (v))] for the purpose of transferring a desired DNA sequence from a plant of a first genotype to a plant of a second genotype.”
These two excerpts from Shen, particularly “repeating steps (b) and (c)” of paragraph 0111, are such that after the first repetition, the “progeny plant” is comparable to “a transgenic plant, or a seed (or T2 plant obtained therefrom) obtained from the transgenic plant, grown from a selected transgenic seed obtained in accordance with the method of claim 30” recited in claim 31.
Conclusion
No claims allowed.
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Any inquiry concerning this communication or earlier communications from the examiner should be directed to YVETTE B TAMUKONG whose telephone number is (571)272-1040. The examiner can normally be reached M-Th 730-5 EST.
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/YVETTE BIH TAMUKONG/ Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/ Supervisory Patent Examiner, Art Units 1661 & 1662
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