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 .
Information Disclosure Statement
Initialed and dated copy of Applicant’s information disclosure statements (IDSs) filed on 04/17/2024 (two documents) are attached to the instant Office action. The submissions are in compliance with the provisions of 37 C.F.R. § 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Status of the Claims
Claims 1-17, 19-29, 31-33 and 35 as filed on 03/27/2024 are pending and are examined in this Office action.
Claim Objections
Claim 31 is objected to for reciting in line 1 the transitional term “comprising” followed by method steps; cf. claim 1. It is suggested to insert the phrase ---, the method--- or ---, said method--- before “comprising” in line 1 of claim 31.
Claim 17 recites in line 3 the misspelled term “trichostain A (TSA)”, which should be replaced with ---trichostatin A (TSA)---.
Appropriate correction is required.
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.
The following is a quotation of the second paragraph of 35 U.S.C. 112:
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.
Indefiniteness
Claims 31-33 are rejected under 35 U.S.C. § 112(b), as being indefinite for failing to particularly point out and distinctly claim the subject matter which 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.
The phrases “increased spectrum of light”, “increased heat treatment”, and “cold-shock treatment” in claim 31 are relative phrases, which render the claim indefinite. The specification does not support the properties of these relative phrases, and it is not clear what exactly the recited increases and treatment are referring to, and how the conditions are effected; in the claimed methods of pre-treatment of seedlings. Is increased spectrum of light a wider range of light wavelengths (within the visible spectrum or extending into the invisible parts of the electromagnetic spectrum)? Ot is it increased frequency and/or energy of the light waves, or any combinations thereof? Is increased heat treatment an increase in temperature, and increase in the time of exposure to heat, a decrease in relative humidity, or any combinations thereof? Are(n’t) these plant species-specific? Dependent claims 32-33 address only the heat treatment aspect, but they do not cure the remaining relative phrase(s) limitations.
In the interest of compact prosecution, the claims are nonetheless examined.
Claim Rejections - 35 USC § 102
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 1-2, 10, 19-21, and 29 are rejected under 35 U.S.C. § 102(a)(1) as being anticipated by WU (Wu et al., United States Patent No. 8,404,930 B2, issued March 26, 2013).
The claims are drawn to a method of pre-treating a seedling with a chemical compound, which comprises contacting the seedling with the chemical compound and isolating leaf explants from the seedling for transformation, wherein the transformation frequency of the leaf explants from the pre-treated seedling is higher than the transformation frequency of leaf explants from seedlings not treated with the chemical compound; and wherein the chemical compound is 2,4-dichlorophenoxyacetic acid (2,4-D), copper, or 6-benzylaminopurine (BAP).
WU teaches methods for improving monocot transformation, such as maize and sorghum, using a combination of media (i.e., chemical conditions) and light (i.e., non-chemical conditions) to achieve increased efficiency of monocot transformation and increased callus initiation frequencies (entire document; see Title, Abstract, for example).
WU teaches a combined media system and methods for the tissue culture and for the transformation of monocots, as methods for increasing transformation efficiency /frequency, etc. Generally, the methods include culturing a plant cell of an explant in an altered environment (i.e., a method of pre-treating a seedling), for example, by altering the kinds and amount of various plant hormones or metal in the culture medium (cf. instant claim 1) and/or levels and duration of exposure to light (cf. instant claim 31). (col 2, lines 48-56; col. 3, lines 47-57).
WU teaches that the term “explant” includes a plant tissue such as young leaf bases, leaves, green tissue, and shoot apexes (col. 4, lines 2-9).
WU specifically teaches (col. 5, section A) preculturing the plant cells prior to transformation. WU teaches that culturing includes, but is not limited to, the steps of preculturing, co-cultivating, resting, selecting, regenerating, or rooting of the transformed monocot plant cells or combinations thereof. Prior to introducing into the monocot plant cells the nucleic acid, the monocot plant cells, such as immature embryo, may be precultured (i.e., a method of pre-treating a seedling). "Precultured" or "preculturing" means culturing the cells or tissues in an appropriate preculture medium to support plant tissue growth prior to the introduction of a nucleic acid, for example, via Agrobacterium-infection, electroporation, or particle bombardment. Preculturing the plant cells may be performed using any method and media known to one ordinarily skilled in the art. Such protocols may vary depending on the species of plant being transformed and the transformation technique employed (col. 5, lines 22-39). Examples of suitable medium for preculturing monocot plant cells include, but are not limited to, MS-based media, LS salts- based media, CM4C, M7, or N6-based media, PHI-T, DBCl, DBC2, and DBC3 which may be supplemented as desired (col. 5, lines 42-52). The pre-culturing step may be performed for as little as a few hours, such as 1 or 2, or for an extended period of time, for example, from about 1 to 7 days or more. Preculturing in combination with the resting and/or selecting steps may increase the transformation frequency/efficiency (col. 5, lines 58-62).
PHI-T includes PHI-I with reducing sucrose to 20 g/L (cf. instant claim 19) and glucose to 10 g/L, increasing 2,4-D to 2 mg/L, adding 0.5 g/L 50 MES buffer, 0.7 g/L L-proline, 10 mg/L ascorbic acid, 100 μM acetosyringone and 8 g/L agar, pH 5.8 (col. 16, lines 13-16) (cf. instant claim 21).
DBCl contains MS salts (4.3 g/L) plus maltose (30 g/L) (cf. instant claim 19); thiamine-HCl (10 mL/L (0.1 mg/mL); myo-inositol (0.25 g/L); N-Z-amine-A (1 g/L)(casein Hydrolysate); praline (0.69 g/L); CuSO4 (4.9 μM); 2,4-D (2.0 mg/L); BAP (0.01 35 mg/L) (col. 16, lines 31-35) (cf. instant claim 10).
DBC2 contains MS salts (4.3 g/L) plus maltose (30 g/L) (cf. instant claim 19); thiamine-HCl (10 mL/L (0.2 mg/mL); myo-inositol (0.25 g/L); N-Z-amine-A (1 g/L) (casein hydrolysate); praline 40 (0.69 g/L); CuSO4 (4.9 μM); 2,4-D (2.0 mg/L); BAP (0.1 mg/L) (col. 16, lines 37-42) (cf. instant claim 10).
DBC3 contains MS salts (4.3 g/L) plus maltose (30 g/L) (cf. instant claim 19); thiamine-RC! (10 mL/L (0.2 mg/mL); myo-inositol (0.25 45 g/L); N-Z-amine-A (1 g/L) (casein hydrolysate); praline (0.69 g/L); CuSO4 (4.9 μM); 2,4-D (1.0 mg/L); BAP (0.5 mg/L) (col. 16, lines 43-47) (cf. instant claim 10).
WU teaches that the media may include an additive such as copper (cf. instant claim 20) and an auxin (col. 6, lines 36-37). Exemplary auxins may include 2,4-dichlorophenoxyacetic acid (2,4-D) (col. 6, lines 53-55) (cf. instant claims 2, 29). WU also teaches that the culture medium includes cytokinin, where exemplary cytokinins include 6-benzylaminopurine (BAP) (col. 7, lines 9-10) (cf. instant claim10). In Example 14 (cols. 30-31), WU teaches that 1 μM and 5 μM (and above) copper increased transformation efficiency in maize.
Accordingly, WU anticipated the claimed invention.
Claims 1, 19, 21, 31 and 35 are rejected under 35 U.S.C. § 102(a)(1) as being anticipated by ZHANG (Zhang et al., 2013, Enhanced Agrobacterium-mediated transformation efficiencies in monocot cells is associated with attenuated defense responses, Plant Mol. Biol. 81: 273-286).
The claims are drawn to a method of pre-treating a seedling, which comprises exposing the seedling to a cold-shock treatment, wherein the transformation frequency of the leaf explants from the pre-treated seedling is higher than the transformation frequency of leaf explants from an unexposed seedling; and wherein the seedling is further contacted with a chemical compound.
ZHANG teaches that enhanced Agrobacterium-mediated transformation efficiencies in monocot cells are associated with attenuated defense responses (entire document; see Title, Abstract, for example).
ZHANG teaches that the utilization of culture conditions associated with an attenuation of defense responses in monocot plant cells led to highly improved Agrobacterium-mediated transformation efficiencies (Abstract). The removal of myo-inositol from the callus culture media in combination with a cold shock pretreatment (cf. instant claim 31) and the addition of L-Gln (glutamine) (cf. instant claim 21) prior to and during Agrobacterium-infection resulted in about 84 % of the treated calluses being stably transformed. The omission of myo-inositol from the callus culture media was associated with the failure of certain pathogenesis related genes to be induced after Agrobacterium infection. The addition of a cold shock and supplemental Gln appeared to have synergistic effects on infection and transformation efficiencies (Abstract).
ZHANG teaches that, by using Agrobacterium as a vehicle for genetic transformation, an array of plant defense responses will be elicited during the course of infection (page 274, left col.).
ZHANG specifically teaches the application of cold shock prior to Agrobacterium infection, and addition of 100 µM L-Gln (Gln+) to the solution prior to and during the infection (Fig. 1) (i.e., simultaneously or sequentially; cf. instant claim 35) (page 275, left col.). Cold shock treatment was applied by immersing the calluses in 3 % maltose (i.e., chemical compound) and placing the samples on ice for 20 min prior to infection (Fig. 1a) (page 275, left col.). The addition of L-Gln (i.e., chemical compound) to the maltose solution had a positive effect on transformation frequencies (Id.).
Accordingly, ZHANG anticipated the claimed invention.
It is also noted, with respect to all of the art rejections described above, that the instantly claimed higher transformation frequency of the leaf explants would be inherent to the compositions taught by the cited art, which comprise the recited structure(s) that are required for the claimed function(s). Applicants are reminded that the express, implicit, and inherent disclosures of a prior art reference may be relied upon in the rejection of claims under 35 U.S.C. § 102 or § 103. “The inherent teaching of a prior art reference, a question of fact, arises both in the context of anticipation and obviousness.” In re Napier, 55 F.3d 610, 613, 34 USPQ2d 1782, 1784 (Fed. Cir. 1995) (affirmed a 35 U.S.C. § 103 rejection based in part on inherent disclosure in one of the references). See also In re Grasselli, 713 F.2d 731, 739, 218 USPQ 769, 775 (Fed. Cir. 1983). See MPEP § 2112.
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 of this title, 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 32-33 are rejected under 35 U.S.C. § 103 as being unpatentable over WU (Wu et al., United States Patent No. 8,404,930 B2, issued March 26, 2013) in view of GUREL (Gurel et al., 2009, Efficient, reproducible Enhanced Agrobacterium-mediated transformation of sorghum using heat treatment of immature embryos, Plant Cell Reports 28: 429-444).
The claims are drawn to a method of pre-treating a seedling, which comprises exposing the seedling to an increased spectrum of light, increased heat treatment, or a cold-shock treatment, wherein the transformation frequency of the leaf explants from the pre-treated seedling is higher than the transformation frequency of leaf explants from an unexposed seedling; wherein the heat treatment is 45ºC for 3 hours or 37ºC for 17 hours at 50% relative humidity.
WU teaches methods for improving monocot transformation, such as maize and sorghum, using a combination of media (i.e., chemical conditions) and light (i.e., non-chemical conditions) to achieve increased efficiency of monocot transformation and increased callus initiation frequencies (entire document; see Title, Abstract, for example).
WU teaches a combined media system and methods for the tissue culture and for the transformation of monocots, as methods for increasing transformation efficiency /frequency, etc. Generally, the methods include culturing a plant cell of an explant in an altered environment (i.e., a method of pre-treating a seedling), for example, by altering the kinds and amount of various plant hormones or metal in the culture medium and/or levels and duration of exposure to light. (col 2, lines 48-56; col. 3, lines 47-57).
WU teaches that the term “explant” includes a plant tissue such as young leaf bases, leaves, green tissue, and shoot apexes (col. 4, lines 2-9).
WU specifically teaches (col. 5, section A) preculturing the plant cells prior to transformation. WU teaches that culturing includes, but is not limited to, the steps of preculturing, co-cultivating, resting, selecting, regenerating, or rooting of the transformed monocot plant cells or combinations thereof. Prior to introducing into the monocot plant cells the nucleic acid, the monocot plant cells, such as immature embryo, may be precultured (i.e., a method of pre-treating a seedling). "Precultured" or "preculturing" means culturing the cells or tissues in an appropriate preculture medium to support plant tissue growth prior to the introduction of a nucleic acid, for example, via Agrobacterium-infection, electroporation, or particle bombardment. Preculturing the plant cells may be performed using any method and media known to one ordinarily skilled in the art. Such protocols may vary depending on the species of plant being transformed and the transformation technique employed (col. 5, lines 22-39). Examples of suitable medium for preculturing monocot plant cells include, but are not limited to, MS-based media, LS salts- based media, CM4C, M7, or N6-based media, PHI-T, DBCl, DBC2, and DBC3 which may be supplemented as desired (col. 5, lines 42-52). The pre-culturing step may be performed for as little as a few hours, such as 1 or 2, or for an extended period of time, for example, from about 1 to 7 days or more. Preculturing in combination with the resting and/or selecting steps may increase the transformation frequency/efficiency (col. 5, lines 58-62).
PHI-T includes PHI-I with reducing sucrose to 20 g/L and glucose to 10 g/L, increasing 2,4-D to 2 mg/L, adding 0.5 g/L 50 MES buffer, 0.7 g/L L-proline, 10 mg/L ascorbic acid, 100 μM acetosyringone and 8 g/L agar, pH 5.8 (col. 16, lines 13-16).
DBCl contains MS salts (4.3 g/L) plus maltose (30 g/L); thiamine-HCl (10 mL/L (0.1 mg/mL); myo-inositol (0.25 g/L); N-Z-amine-A (1 g/L)(casein Hydrolysate); praline (0.69 g/L); CuSO4 (4.9 μM); 2,4-D (2.0 mg/L); BAP (0.01 35 mg/L) (col. 16, lines 31-35).
DBC2 contains MS salts (4.3 g/L) plus maltose (30 g/L); thiamine-HCl (10 mL/L (0.2 mg/mL); myo-inositol (0.25 g/L); N-Z-amine-A (1 g/L) (casein hydrolysate); praline 40 (0.69 g/L); CuSO4 (4.9 μM); 2,4-D (2.0 mg/L); BAP (0.1 mg/L) (col. 16, lines 37-42).
DBC3 contains MS salts (4.3 g/L) plus maltose (30 g/L); thiamine-RC! (10 mL/L (0.2 mg/mL); myo-inositol (0.25 45 g/L); N-Z-amine-A (1 g/L) (casein hydrolysate); praline (0.69 g/L); CuSO4 (4.9 μM); 2,4-D (1.0 mg/L); BAP (0.5 mg/L) (col. 16, lines 43-47).
WU teaches that the media may include an additive such as copper and an auxin (col. 6, lines 36-37). Exemplary auxins may include 2,4-dichlorophenoxyacetic acid (2,4-D) (col. 6, lines 53-55). WU also teaches that the culture medium includes cytokinin, where exemplary cytokinins include 6-benzylaminopurine (BAP) (col. 7, lines 9-10). In Example 14 (cols. 30-31), WU teaches that 1 μM and 5 μM (and above) copper increased transformation efficiency in maize.
WU does not explicitly teach all of the instantly claimed method steps (e.g., heat treatment at 45ºC for 3 hours or 37ºC for 17 hours at 50% relative humidity. However, such claimed compositions and methods would have been prima facie obvious to a person of ordinary skill in the art at the time of filing the instant application for the following reasons.
GUREL teaches efficient, reproducible Agrobacterium-mediated transformation
of sorghum using heat treatment of immature embryos (entire document; see Title, Abstract, for example). A number of parameters related to Agrobacterium-mediated infection were tested to optimize transformation frequencies of sorghum (Abstract).
GUREL teaches incubation of the isolated sorghum embryos at 37, 40, 43 and 46ºC in a carefully controlled water bath; either before inoculation with A. tumefaciens or before centrifugation that preceded Agrobacterium inoculation. Duration of the heat treatment was either 3 min for all temperatures or 5, 10 and 30 min for 43ºC with and without centrifugation (page 431, left col.).
In Table 1 on page 434, GUREL teaches the effect of cold and centrifugation treatments on transient and stable transformation frequency of sorghum.
Given the teachings of WU and GUREL as described above, it would have been prima facie obvious to a person of ordinary skill in the art at the time of filing the instant application to use and to modify the compositions and methods taught by WU and GUREL, and this would result in the Applicants’ invention; with a reasonable expectation of success, and without any surprising results. Obviously, one of ordinary skill in the art would have been motivated to do so for the purpose of improving plant transformation efficiency, as taught by WU and GUREL.
Although WU and GUREL do not explicitly reduce to practice a heat treatment of 45ºC for 3 hours or 37ºC for 17 hours at 50% relative humidity, these particular embodiments would be considered routine optimization and a design choice that would be readily apparent to one of ordinary skill in the art. The cited art teaches using different temperatures and different heating times, culturing a plant cell of an explant in an altered environment, and in general altering the levels and duration of exposure. Thus the recited limitations would be desirable optimization of the methods and a design choice that would be readily apparent to one of ordinary skill in the art, absent evidence to the contrary.
Claim 15 is rejected under 35 U.S.C. § 103 as being unpatentable over WU (Wu et al., United States Patent No. 8,404,930 B2, issued March 26, 2013) as applicable to claim 1 above (see the rejection under 35 USC § 102) in view of NAKANO (Nakano et al., 2018, FPX is a novel chemical inducer that promotes callus formation and shoot regeneration in plants, Plant & Cell Physiology 59(8): 1555-1567).
The claim is drawn to a method of pre-treating a seedling with a chemical compound, which comprises contacting the seedling with the chemical compound and isolating leaf explants from the seedling for transformation, wherein the transformation frequency of the leaf explants from the pre-treated seedling is higher than the transformation frequency of leaf explants from seedlings not treated with the chemical compound; and wherein the chemical compound is fipexide.
Claim 15 is dependent on claim 1.
WU does not explicitly teach that the chemical compound is fipexide.
However, NAKANO teaches that fipexide (FPX) is a novel chemical inducer that promotes callus formation and shoot regeneration in plants (entire document; see Title, Abstract, for example). Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency; ethylene is well known as an inhibitor of transformation efficiency (Abstract; Conclusion, page 5, left col.).
NAKANO teaches that FPX promotes callus formation and shoot regeneration, and facilitates Agrobacterium transformation of monocots and tree species. FPX is able to induce tree callus that has shoot regeneration ability and the capacity for Agrobacterium-mediated transformation. Furthermore, four days after germination, 45 and 100mM FPX induced callus from mature Brachypodium seeds ( (page 1560, right col.).
Given the teachings of WU and NAKANO as described above, it would have been prima facie obvious to a person of ordinary skill in the art at the time of filing the instant application to use and to modify the compositions and methods taught by WU and NAKANO, and this would result in the Applicants’ invention; with a reasonable expectation of success, and without any surprising results. Obviously, one of ordinary skill in the art would have been motivated to do so for the purpose of improving plant transformation efficiency, as taught by WU and NAKANO.
Claims 22-23 are rejected under 35 U.S.C. § 103 as being unpatentable over WU (Wu et al., United States Patent No. 8,404,930 B2, issued March 26, 2013) as applicable to claim 1 above (see the rejection under 35 USC § 102) in view of NONAKA (Nonaka and Ezura, 2014, Plant-Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer, Frontiers in Plant Science vol. 5, Article 681: 1-7).
The claims are drawn to a method of pre-treating a seedling with a chemical compound, which comprises contacting the seedling with the chemical compound and isolating leaf explants from the seedling for transformation, wherein the transformation frequency of the leaf explants from the pre-treated seedling is higher than the transformation frequency of leaf explants from seedlings not treated with the chemical compound; and wherein the chemical compound is an ethylene inhibitor which is silver nitrate or the ethylene competitive inhibitor norbonadiene.
Claims 22-23 are dependent on claim 1.
WU does not explicitly teach that the chemical compound is an ethylene inhibitor which is silver nitrate or the ethylene competitive inhibitor norbonadiene.
However, NONAKA teaches that the plant-Agrobacterium interaction is mediated by ethylene (entire document; see Title, Abstract, for example). Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency; ethylene is well known as an inhibitor of transformation efficiency (Abstract; Conclusion, page 5, left col.).
NONAKA teaches that reducing ethylene production during co-cultivation using ethylene biosynthesis inhibitors such as AVG or suppressing plant ethylene perception by adding silver ions (i.e., silver nitrate; cf. instant claim 22) to the tissue culture medium has improved the transformation efficiency in melons, cauliflowers, apricots, apple trees, and bottle gourds. The stable transformation frequency was also increased in the Arabidopsis thaliana ethylene-insensitive mutants etr1-1 and etr1-2 (page 2, left col.). In addition, plants treated with inhibitors of ethylene synthesis or perception, such as aminoethoxyvinylglycine (AVG), and ethylene-insensitive Never-ripe mutants (tomato) suppress crown gall growth (Id.).
Given the teachings of WU and NONAKA as described above, it would have been prima facie obvious to a person of ordinary skill in the art at the time of filing the instant application to use and to modify the compositions and methods taught by WU and NONAKA, and this would result in the Applicants’ invention; with a reasonable expectation of success, and without any surprising results. Obviously, one of ordinary skill in the art would have been motivated to do so for the purpose of improving plant transformation efficiency, as taught by WU and NONAKA.
Although WU and NONAKA do not explicitly reduce to practice the use of norbornadiene (cf. instant claim 23), this particular embodiment would be considered routine optimization and a design choice that would be readily apparent to one of ordinary skill in the art. It is known in the art that norbornadiene (NBD) acts as an ethylene action inhibitor, which is useful for studies like manipulating growth or influencing transformation efficiency.
Claims 3, 12 and 28-29 are rejected under 35 U.S.C. § 103 as being unpatentable over WU (Wu et al., United States Patent No. 8,404,930 B2, issued March 26, 2013) as applicable to claim 1 above (see the rejection under 35 USC § 102) in view of DAN (Dan et al., 2014, Novel compounds that enhance Agrobacterium-mediated plant transformation by mitigating oxidative stress, Plant Cell Reports 34: 291-309).
The claims are drawn to a method of pre-treating a seedling with a chemical compound, which comprises contacting the seedling with the chemical compound and isolating leaf explants from the seedling for transformation, wherein the transformation frequency of the leaf explants from the pre-treated seedling is higher than the transformation frequency of leaf explants from seedlings not treated with the chemical compound; and wherein the chemical compound is cysteine, caffeine or diphenyleneiodonium (DPI).
Claims 3, 12 and 28-29 are dependent on claim 1.
WU does not explicitly teach that the chemical compound is cysteine, caffeine or DPI.
However, DAN teaches novel compounds that enhance Agrobacterium-mediated plant transformation by mitigating oxidative stress in transformed MicroTom plants (entire document; see Title, Abstract, for example). Agrobacterium caused tissue browning leading to subsequent cell death. Without an antioxidant, tissue browning was severe and associated with low stable transformation frequency (Abstract).
DAN teaches that Agrobacterium causes oxidative stress leading to browning and death of its transformed cells and anti-oxidative compounds reduce tissue browning and cell death of transformed cells and thus increase stable transformation frequency by mitigating oxidative stress. DAN selected a group of new candidate compounds predicted to have the potential to reduce tissue browning and to increase transient expression and stable transformation, based on their demonstrated ability to mitigate ROS (i.e., reactive oxygen species) accumulation in the plant defense response (i.e., ROS scavenger) (page 294, left col.). In Figure 1 on page 294 DAN describes the use of cysteine (cf. instant claim 3). In Figure 6 on page 302 and in Table 1 on page 293 DAN describes the use of caffeic acid (i.e. caffeine) (cf. instant claim 12) and diphenyleneiodonium chloride (i.e., DPI) (cf. instant claims 28-29).
It is also generally known in the art that the accumulation of latent defense proteins including ROS scavenging enzymes results in enhanced stress responses (which positively correlates with higher transformation frequency).
DAN teaches that ROS balance in root meristems involves interactions with other plant growth regulators. Redox gradients may underlie gradients in various activities along the root axis, including auxin regulation of root development (page 307, left col.).
Given the teachings of WU and DAN as described above, it would have been prima facie obvious to a person of ordinary skill in the art at the time of filing the instant application to use and to modify the compositions and methods taught by WU and DAN, and this would result in the Applicants’ invention; with a reasonable expectation of success, and without any surprising results. Obviously, one of ordinary skill in the art would have been motivated to do so for the purpose of improving plant transformation efficiency, as taught by WU and DAN.
Claim 26 is rejected under 35 U.S.C. § 103 as being unpatentable over WU (Wu et al., United States Patent No. 8,404,930 B2, issued March 26, 2013) in view of DAN (Dan et al., 2014, Novel compounds that enhance Agrobacterium-mediated plant transformation by mitigating oxidative stress, Plant Cell Reports 34: 291-309) as applicable to claims 1, 3, 12 and 28-29 above (see also the rejection under 35 USC § 102) and further in view of KHAN (Khan et al., 2015, Salicylic acid-induced abiotic stress tolerance and underlying mechanisms in plants, Frontiers in Plant Science 6: 462, 1-17).
The claim is drawn to a method of pre-treating a seedling with a chemical compound, which comprises contacting the seedling with the chemical compound and isolating leaf explants from the seedling for transformation, wherein the transformation frequency of the leaf explants from the pre-treated seedling is higher than the transformation frequency of leaf explants from seedlings not treated with the chemical compound; and wherein the chemical compound is salicylic acid (SA).
WU and DAN teach higher transformation frequency based on ROS mitigation in the plant defense response, but they do not explicitly teach that the chemical compound is salicylic acid.
However, KHAN teaches the salicylic acid-induced abiotic stress tolerance and underlying mechanisms in plants (entire document; see Title, Abstract, for example). KHAN teaches the involvement of salicylic acid in ROS-signaling and the modulation of antioxidants (section bridging pages 8-9). Both endogenous and exogenous SA was evidenced to play roles in antioxidant metabolism and have a tight control over cellular ROS (page 8, right col., last paragraph). KHAN teaches that the involvement of SA in the modulation of antioxidant metabolism has been widely reported to control plant-tolerance to major abiotic stresses (page 9, left col.).
Given the teachings of WU, DAN, and KHAN as described above, it would have been prima facie obvious to a person of ordinary skill in the art at the time of filing the instant application to use and to modify the compositions and methods taught by WU, DAN, and KHAN, and this would result in the Applicants’ invention; with a reasonable expectation of success, and without any surprising results. Obviously, one of ordinary skill in the art would have been motivated to do so for the purpose of improving plant transformation efficiency, as taught by WU and DAN, and using the SA teachings of KHAN.
Claims 16 and 29 are rejected under 35 U.S.C. § 103 as being unpatentable over WU (Wu et al., United States Patent No. 8,404,930 B2, issued March 26, 2013) as applicable to claim 1 above (see the rejection under 35 USC § 102) in view of ZHAO (Zhao et al., 2020, Improving T-DNA transfer to Tamarix hispida by adding chemical compounds during Agrobacterium tumefaciens culture, Frontiers in Plant Science vol. 5, Article 681: 1-7).
The claims are drawn to a method of pre-treating a seedling with a chemical compound, which comprises contacting the seedling with the chemical compound and isolating leaf explants from the seedling for transformation, wherein the transformation frequency of the leaf explants from the pre-treated seedling is higher than the transformation frequency of leaf explants from seedlings not treated with the chemical compound; and wherein the chemical compound is 5-azacytidine.
Claims 16 and 29 are dependent on claim 1.
WU does not explicitly teach that the chemical compound is 5-azacytidine.
However, ZHAO teaches the improvement of T-DNA transfer to Tamarix hispida by adding chemical compounds during Agrobacterium tumefaciens culture (entire document; see Title, Abstract, for example).
ZHAO investigated the factors that affect the genetic transformation efficiency during Agrobacterium culture using Tamarix hispida as transgenic plant material. Agrobacterium treatment with spermidine (Spe), azacitidine (5-AzaC), dithiothreitol (DTT), or acetosyringone (AS) alone all significantly improved the efficiency of T-DNA transfer. Treatment with 5-AzaC reduced DNA methylation in Agrobacterium to induce the expression of virulence (vir) family genes (Abstract).
ZHAO teaches that azacytidine is a DNA demethylating agent that can reduce or inhibit DNA methylation. Agrobacterium cells treated with 5-AzaC showed significantly increased transient expression efficiency, and AzaC also inhibits the methylation-dependent inactivation of the transformed gene. These results suggested that 5-AzaC treatment could improve the genetic transformation efficiency (page 6, right col., last paragraph).
Given the teachings of WU and ZHAO as described above, it would have been prima facie obvious to a person of ordinary skill in the art at the time of filing the instant application to use and to modify the compositions and methods taught by WU and ZHAO, and this would result in the Applicants’ invention; with a reasonable expectation of success, and without any surprising results. Obviously, one of ordinary skill in the art would have been motivated to do so for the purpose of improving plant transformation efficiency, as taught by WU and ZHAO.
Claim 13 is rejected under 35 U.S.C. § 103 as being unpatentable over WU (Wu et al., United States Patent No. 8,404,930 B2, issued March 26, 2013) as applicable to claim 1 above (see the rejection under 35 USC § 102) in view of HWANG (Hwang et al., 2020, Agrobacterium-produced and exogenous cytokinin-modulated Agrobacterium-mediated plant transformation, Molecular Plant Pathology 11: 677-690).
The claim is drawn to a method of pre-treating a seedling with a chemical compound, which comprises contacting the seedling with the chemical compound and isolating leaf explants from the seedling for transformation, wherein the transformation frequency of the leaf explants from the pre-treated seedling is higher than the transformation frequency of leaf explants from seedlings not treated with the chemical compound; and wherein the chemical compound is an auxin agonist selected from the group consisting of quinclorac, RubNeddins1 (RN1), RubNeddins2 (RN2), RubNeddins3 (RN3), and RubNeddins4 (RN4).
Claim 13 is dependent on claim 1.
WU does not explicitly teach that the chemical compound is an auxin agonist selected from the group consisting of quinclorac, RubNeddins1 (RN1), RubNeddins2 (RN2), RubNeddins3 (RN3), and RubNeddins4 (RN4).
However, HWANG teaches that a previous study has demonstrated that auxin pretreatment of petiole explants from transformation-recalcitrant Arabidopsis ecotypes significantly enhances their susceptibility to transformation. HWANG’s study demonstrated that exogenous auxin treatments before Agrobacterium infections could have positive effects on the subsequent genetic transformations (page 678, left col.).
Given the teachings of WU and HWANG as described above, it would have been prima facie obvious to a person of ordinary skill in the art at the time of filing the instant application to use and to modify the compositions and methods taught by WU and HWANG, and this would result in the Applicants’ invention; with a reasonable expectation of success, and without any surprising results. Obviously, one of ordinary skill in the art would have been motivated to do so for the purpose of improving plant transformation efficiency, as taught by WU and HWANG.
Although WU and HWANG do not explicitly reduce to practice the use of an auxin agonist selected from the group consisting of quinclorac, RubNeddins1 (RN1), RubNeddins2 (RN2), RubNeddins3 (RN3), and RubNeddins4 (RN4), these particular embodiments would be considered routine optimization and a design choice that would be readily apparent to one of ordinary skill in the art; absent evidence to the contrary. It is known in the at that an auxin agonist is a molecule that mimics the function of auxin by activating the same signaling pathways to influence plant growth and development.
Claim 17 is rejected under 35 U.S.C. § 103 as being unpatentable over WU (Wu et al., United States Patent No. 8,404,930 B2, issued March 26, 2013) as applicable to claim 1 above (see the rejection under 35 USC § 102) in view of CHOI (Choi et al., 2020, TSA promotes CRISPR/Cas9 editing efficiency and expression of cell division-related genes from plant protoplasts, International Journal of Molecular Sciences 22, 7817, pp 1-15).
The claim is drawn to a method of pre-treating a seedling with a chemical compound, which comprises contacting the seedling with the chemical compound and isolating leaf explants from the seedling for transformation, wherein the transformation frequency of the leaf explants from the pre-treated seedling is higher than the transformation frequency of leaf explants from seedlings not treated with the chemical compound; and wherein the chemical compound is trichostatin A (TSA).
Claim 17 is dependent on claim 1.
WU does not explicitly teach that the chemical compound is trichostatin A.
However, CHOI teaches that TSA promotes CRISPR/Cas9 editing efficiency and expression of cell division-related genes from plant protoplasts (entire document; see Title, Abstract, for example). Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 µM). Chromatin accessibility with TSA treatments was higher than that of DMSO treatment (control). Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. This could be applied for the development of useful genome-edited crops (Abstract).
Given the teachings of WU and CHOI as described above, it would have been prima facie obvious to a person of ordinary skill in the art at the time of filing the instant application to use and to modify the compositions and methods taught by WU and CHOI, and this would result in the Applicants’ invention; with a reasonable expectation of success, and without any surprising results. Obviously, one of ordinary skill in the art would have been motivated to do so for the purpose of improving plant transformation efficiency, as taught by WU and CHOI. As taught by CHOI, pretreating plants with a histone deacetylase (HDAC) inhibitor, such as trichostatin A (TSA) can increase transformation efficiency by making the chromatin more accessible. This process opens up the chromatin structure, which may allow greater access for gene editing tools like CRISPR/Cas9 to target DNA and improve the efficiency of the DNA repair machinery. It can also enhance callus proliferation and the overall expression of genes involved in cell division.
Claims 4-6 are rejected under 35 U.S.C. § 103 as being unpatentable over WU (Wu et al., United States Patent No. 8,404,930 B2, issued March 26, 2013) as applicable to claim 1 above (see the rejection under 35 USC § 102) in view of THAKUR (Thakur et al., 2006, Regulation of growth of Lilium plantlets in liquid medium by application of paclobutrazol or ancymidol, for its amenability in a bioreactor system: growth parameters, Molecular Plant Pathology 11: 677-690).
The claims are drawn to a method of pre-treating a seedling with a chemical compound, which comprises contacting the seedling with the chemical compound and isolating leaf explants from the seedling for transformation, wherein the transformation frequency of the leaf explants from the pre-treated seedling is higher than the transformation frequency of leaf explants from seedlings not treated with the chemical compound; and wherein the chemical compound is paclobutrazol or ancymidol at a concentration of about 1 mg/l to about 2 mg/l.
Claims 4-6 are dependent on claim 1.
WU does not explicitly teach that the chemical compound is paclobutrazol or ancymidol at a concentration of about 1 mg/l to about 2 mg/l.
However, THAKUR teaches the regulation of growth of Lilium plantlets in liquid medium by application of paclobutrazol or ancymidol (entire document; see Title, Abstract, for example).
THAKUR teaches that a significant increase in leaf chlorophyll, epicuticular wax, plant dry weight and bulb starch contents were found in plantlets treated with the growth retardants paclobutrazol or ancymidol. A similar increase in the number of leaves, roots and bulbs was also noted. Treatment of plantlets with 3.4 µM paclobutrazol was found to be the best (Abstract).
THAKUR teaches liquid MS media supplemented with 1.7 µM – 17 µM paclobutrazol (PBZ) or 1.7 µM – 17 µM ancymidol (ANC) (Materials and methods, page 383, left col.). For both ANC and PBZ, best growth characteristics were obtained at 1.7 and 3.4 µM, and the observations for the various growth parameters of the plantlets and their ex vitro survival after 40 days of culture are presented in Fig.1 (Results, page 384, right col.).
Given the teachings of WU and THAKUR as described above, it would have been prima facie obvious to a person of ordinary skill in the art at the time of filing the instant application to use and to modify the compositions and methods taught by WU and THAKUR, and this would result in the Applicants’ invention; with a reasonable expectation of success, and without any surprising results. Obviously, one of ordinary skill in the art would have been motivated to do so for the purpose of improving plant transformation efficiency, as taught by WU, using the teachings of THAKUR for generating robust plantlets capable of successful ex vitro transfer.
Although WU and THAKUR do not explicitly reduce to practice the use of ancymidol at a concentration of about 1 mg/l to about 2 mg/l, this particular embodiment would be considered routine optimization and a design choice that would be readily apparent to one of ordinary skill in the art; absent evidence to the contrary.
Claims 7-8 and 11 are rejected under 35 U.S.C. § 103 as being unpatentable over WU (Wu et al., United States Patent No. 8,404,930 B2, issued March 26, 2013) as applicable to claim 1 above (see the rejection under 35 USC § 102) in view of TSUDA (Tsuda et al., 2012, An efficient Agrobacterium-mediated transient transformation of Arabidopsis, The Plant Journal 69: 713-719).
The claims are drawn to a method of pre-treating a seedling with a chemical compound, which comprises contacting the seedling with the chemical compound and isolating leaf explants from the seedling for transformation, wherein the transformation frequency of the leaf explants from the pre-treated seedling is higher than the transformation frequency of leaf explants from seedlings not treated with the chemical compound; and wherein the chemical compound is an inhibitor of abscisic acid biosynthesis selected from the group consisting of norflurazon, fluridone, diflufenican, abamine, and nordihydroguaiaretic acid (NDGA), an abscisic acid antagonist selected from the group consisting of AA1 [1-[(4-benzylpiperazin-1-yl)methyl]-2-sulfanylidene-1H,2H,5H,6H,7H,8H-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-5-one], AS6 [3′-hexylsulfanyl-ABA], DFPM ([5-(3,4-Dichlorophenyl) furan-2-yl]-piperidin-1-ylmethanethione), and RK460, or an anti-cytokinin selected from the group consisting of S-4893 [a 4-phenylquinazoline derivative], 4-phenylquinazoline, 6-(2,5-Dihydroxybenzylamino) purine (LGR-991), and 2-hydroxy-3-methyl-benzyladenine.
Claims 7-8 and 11 are dependent on claim 1.
WU does not explicitly teach that the chemical compound is an inhibitor of abscisic acid biosynthesis, or an abscisic acid antagonist.
However, TSUDA teaches an efficient Agrobacterium-mediated transient transformation of Arabidopsis (entire document; see Title, Abstract, for example).
TSUDA cites various other references, teaching that plant hormones, such as cytokinin, auxin, and abscisic acid, interfere with Agrobacterium infection (page 714, left col.).
Given the teachings of WU and TSUDA as described above, it would have been prima facie obvious to a person of ordinary skill in the art at the time of filing the instant application to use and to modify the compositions and methods taught by WU and TSUDA, and this would result in the Applicants’ invention; with a reasonable expectation of success, and without any surprising results. Obviously, one of ordinary skill in the art would have been motivated to do so for the purpose of improving plant transformation efficiency, as taught by WU, using the teachings of TSUDA that abscisic acid and cytokinin interfere with Agrobacterium infection.
Although WU and TSUDA do not explicitly reduce to practice the use of inhibitor of abscisic acid biosynthesis selected from the group consisting of norflurazon, fluridone, diflufenican, abamine, and nordihydroguaiaretic acid (NDGA), an abscisic acid antagonist selected from the group consisting of AA1 [1-[(4-benzylpiperazin-1-yl)methyl]-2-sulfanylidene-1H,2H,5H,6H,7H,8H-[1,3,4]thiadiazolo[3,2-a][1,3]diazepin-5-one], AS6 [3′-hexylsulfanyl-ABA], DFPM ([5-(3,4-Dichlorophenyl) furan-2-yl]-piperidin-1-ylmethanethione), and RK460, or anti-cytokinin selected from the group consisting of S-4893 [a 4-phenylquinazoline derivative], 4-phenylquinazoline, 6-(2,5-Dihydroxybenzylamino) purine (LGR-991), and 2-hydroxy-3-methyl-benzyladenine, these particular embodiment would be considered routine optimization and a design choice that would be readily apparent to one of ordinary skill in the art; absent evidence to the contrary.
Claim 25 is rejected under 35 U.S.C. § 103 as being unpatentable over WU (Wu et al., United States Patent No. 8,404,930 B2, issued March 26, 2013) as applicable to claim 1 above (see the rejection under 35 USC § 102) in view of NAGIRA (Nagira et al., United States Patent No. 10,550,399 B2, issued February 4, 2020).
The claim is drawn to a method of pre-treating a seedling with a chemical compound, which comprises contacting the seedling with the chemical compound and isolating leaf explants from the seedling for transformation, wherein the transformation frequency of the leaf explants from the pre-treated seedling is higher than the transformation frequency of leaf explants from seedlings not treated with the chemical compound; and wherein the chemical compound is jasmonic acid.
Claim 25 is dependent on claim 1.
WU does not explicitly teach that the chemical compound is jasmonic acid.
However, NAGIRA teaches a plant transformation method using plant growth inhibiting hormone culture (entire document; see Title, Abstract, for example).
NAGIRA teaches and claims (claim 1 of NAGIRA) a method of transforming a plant, which comprises inoculating an Agrobacterium solution comprising jasmonic acid at a final concentration of from 10 to 100 μM and an Agrobacterium carrying a vector comprising a foreign gene into a wound site of a shoot apical meristem of a germinated seed having a shoot, and infecting the shoot apical meristem with the Agrobacterium.
Given the teachings of WU and NAGIRA as described above, it would have been prima facie obvious to a person of ordinary skill in the art at the time of filing the instant application to use and to modify the compositions and methods taught by WU and NAGIRA, and this would result in the Applicants’ invention; with a reasonable expectation of success, and without any surprising results. Obviously, one of ordinary skill in the art would have been motivated to do so for the purpose of improving plant transformation efficiency, as taught by WU and NAGIRA.
It is also noted, with respect to all of the art rejections described above, that the instantly claimed higher transformation frequency of the leaf explants would be inherent to the compositions taught by the cited art, which comprise the recited structure(s) that are required for the claimed function(s). Applicants are reminded that the express, implicit, and inherent disclosures of a prior art reference may be relied upon in the rejection of claims under 35 U.S.C. § 102 or § 103. “The inherent teaching of a prior art reference, a question of fact, arises both in the context of anticipation and obviousness.” In re Napier, 55 F.3d 610, 613, 34 USPQ2d 1782, 1784 (Fed. Cir. 1995) (affirmed a 35 U.S.C. § 103 rejection based in part on inherent disclosure in one of the references). See also In re Grasselli, 713 F.2d 731, 739, 218 USPQ 769, 775 (Fed. Cir. 1983). See MPEP § 2112.
Subject Matter Free of Prior Art
Claims 9, 14, 24, and 27 are deemed free of the prior ar