DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 3/26/2026 has been entered.
The rejection of claim(s) 1 & 3-4, 6-7, 9-11, 13 & 16-18 under 35 U.S.C. 103 as being unpatentable over Velcheva et al (2005). Plant Science. 168: 121-130 in view of McCormick (1991) Plant Tissue Culture Manual B6: 1-9 and Hesami et al (2021) Int. J. Mol. Sci. 22, 5671 is withdrawn in light of Applicant’s amendments.
The rejection of claims 1, 3-4 & 6-18 under 35 U.S.C. 103 as being unpatentable over Martinell et al US 8,357,834 B2 (patented 1/22/2013, hereafter Martinell) in view of Petersen et al US 2021/0071186 A1 is withdrawn in light of Applicant’s amendments.
The rejection of claim 5 under 35 U.S.C. 103 as being unpatentable over Martinell and Petersen, as applied to claims 1, 3-4 & 6-18 above, and further in view of Ouma et al (2004) African Journal of Biotechnology Vol. 3 (6): 313-318 is withdrawn in light of Applicant’s amendments.
The rejection of claim 1 on the ground of nonstatutory double patenting as being unpatentable over claims 1, 10 & 5 of U.S. Patent No. 11,266,086 B2 is withdrawn in light of Applicant’s amendments.
Status of Claims
Claims 1 & 3-12, 14-15, 17-18, 22 & 23 are under examination on the merits.
Claims 19-21 remain withdrawn without traverse.
Claim Objections
Claim 23 is objected to because of the following informalities:
Claim 23 (lines 3-4): “L.” should not be italicized in the Latin species names.
Appropriate correction is required.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim(s) 1, 3, 4, 6-10, 14, 17-18 & 22 are rejected under 35 U.S.C. 103 as being unpatentable over Velcheva et al (2005). Plant Science. 168: 121-130 (published 9/1/2004, hereafter Velcheva) in view of Galan-Avila et al (2021) Industrial Crops & Products. 170. 113691. (available online 6/9/2021, hereafter Galan-Avila) and Zhang et al (2021) Plant Biotechnology Journal. 19: 1979-1987 (published October 2021, hereafter Zhang).
This is a new rejection in light of Applicant’s amendments. Applicant' s arguments filed 3/26/2026 have been fully considered as they pertain to the current rejection, but they are not persuasive.
Claims 1, 3-4, 6-10, 14, & 17 are drawn to a method of transforming an explant comprising excising the explant, introducing an exogenous nucleic acid into the plant, and culturing the explant on a liquid selection medium. Claim 18 is drawn to the method wherein the exogenous nucleic acid encodes a guide RNA. Claim 22 is drawn to the method wherein the seed is a Cannabis seed.
Velcheva teaches a method for transformation of tomato with a GUS reporter gene using Agrobacterium tumefaciens (page 122, left column, paragraph 3-right column, paragraph 3). In this method, seeds were sterilized in NaClO, rinsed in distilled water, and germinated on solidified 1/4 Murashige and Skoog (MS) media after which the middle section of the cotyledons and top edge of the hypocotyls were excised to prepare explants.
After co-cultivation with Agrobacterium, explants were cultured in MS initiation liquid medium comprising kanamycin, timentin, zeatin, and IAA (page 123, left column, paragraph 2). Three weeks after transformation, explants were transferred to solidified medium, then elongated shoots were detached and rooted on MS basal medium. Rooting medium was supplemented with 100 mg/L kanamycin (page 123, left column, paragraph 2).
Velcheva teaches that both cotyledon and hypocotyl explants regenerated shoots and shoots were visible 10 days after transfer to MSI for cotyledon explants (page 124, left column, paragraph 1). Velcheva teaches that transformation efficiency was higher when selection was performed in liquid rather than solid medium at rates of >20% transformation efficiency (page 126, right column, paragraph 3-page 127, left column, paragraph 1; figure 6). Velcheva teaches that regeneration was increased if the regeneration media had both IAA and zeatin (page 128, left column, paragraph 2).
Velcheva teaches that increasing the contact of the surface of the explant with selective media increases selection efficiency and that high levels of non-transgenic escapes and chimeric plants are observed when selection is on solid media (page 121, right column, paragraph 1). Velcheva explicitly teaches that selection and regeneration in liquid culture may significantly reduce the percentage of non-transformed regenerants (page 122, left column, paragraph 2).
Velcheva teaches that co-cultivation of Agrobacterium and explants followed the protocol of McCormick (page 123, left column, paragraph 2) but does not teach the method in full.
Velcheva does not teach a method wherein the seed is a Cannabis, Abelmoschus, Vigna, or Arachis seed or wherein the co-culture medium comprises thidiazuron, the length of time that Agrobacterium are co-cultivated with explants, nor the medium in which co-culture is performed, nor transformation with a guide RNA.
Galan-Avila teaches a method for producing stably transformed Cannabis plants three times faster than other protocols and teaches that transgenic plants were obtained from all explant types evaluated (abstract). Cannabis explants were inoculated and then cultured with semi-solid co-culture medium for 4 days with Agrobacterium tumefaciens; the co-culture medium for at least the cotyledon explants comprised thidiazuron (page 3, left column, paragraph 4; table 1). Explants were then cultured on selective regeneration medium comprising Kanamycin, and at least the cotyledon explants were cultured in medium comprising thidiazuron and alpha naphthaleneacetic acid (table 1). Galan-Avila teaches that kanamycin was a poor selectable marker for C. sativa due to the low albinism rate achieved (page 12, left column, paragraph 1-right column, paragraph 1). The method of Galan-Avila does not comprise a step of callus removal.
Galan-Avila provides images of non-transformed hypocotyl explants having spontaneous roots after three weeks of in vitro culture, at least one of which is at least 8mm according to the provided scale bar, but only a partial segment is captured in the photo (figure 1c). Furthermore, Galan-Avila teaches that within 16 days of initiating hypocotyl in vitro culture, explants that were co-cultivated with A. tumefaciens could be observed with spontaneous rooting of regenerating shoots on selective medium, and approximately one month after culture initiation, leaves from transformed shoots had observable GUS staining patterns (page 9, left column, paragraph 2-right column, paragraph 1). At least one transformed explant can be seen with a root at least 8mm long, according to the provided scale bar, at 16 days after co-culture with Agrobacterium (figure 4 e). However, this image also does not capture the full root length.
Galan-Avila teaches that hypocotyl-derived regenerating shoots cultured in kanamycin were able to exhibit a vigorous growth one month after in vitro culture initiation (page 6, left column, paragraph 2). Galan-Avila teaches transformation rates of greater than 1% for some explants and cultivars (table 6). Galan-Avila teaches that genome editing offers a new avenue for breeding C. sativa (page 12, right column, paragraph 2). In addition, Galan-Avila teaches a motivation to use genetic transformation in C. sativa to develop improved varieties resistant to biotic and abiotic stresses, or with increased yield, but that an efficient transformation protocol for regeneration of transgenic plants is imperative (page 2, left column, paragraph 2). However, Galan-Avila teaches that the method is faster than the method of Zhang et al 2021 for CRISPR/Cas genome editing in C. sativa (page 11, right column, paragraph 1).
Zhang teaches a method of Cannabis genome editing wherein shoots are recovered from regeneration medium 78 days after callus induction (figure 1). Zhang also teaches that transgenic shoots were transferred to soil after a 5-week incubation in root-induction medium (figure 6). Zhang teaches a method of genome editing comprising delivering guide RNA targeting the PDS2 gene fused to the vector pG41sg to immature embryo hypocotyls via infection with the Agrobacterium tumefaciens strain AGL1 (page 1981, left column, paragraph 1).
Before the effective filing date of the instant application, it would have been obvious to one of ordinary skill in the art to substitute a Cannabis seed explant for a tomato seed explant in the method of explant transformation taught by Velcheva. One of ordinary skill in the art would have been motivated to use Cannabis seed, because efficient genetic transformation of C. sativa is imperative for developing improved varieties. In using Cannabis, it would have been obvious to use a co-culture medium comprising thidiazuron (TDZ) as taught by Galan-Avila. One of ordinary skill in the art would have had reasonable expectation of success, because tissue culture of Cannabis, including culture with TDZ, led to a rapid transformation and regeneration protocol according to Galan-Avila. Galan-Avila’s method required no callus removal step, which reads on callusing reduced to the degree that callus no longer needs to be removed from the explants. It is noted that although instant claim 1 recites that callus no longer needs to be removed from the explants, the method of claim 1 does not require that the callus is removed or is not removed.
Galan-Avila does not explicitly teach that the rooted Cannabis explants are greenhouse-ready in fewer than 100 days after introduction of the exogenous nucleic acid. Galan-Avila teaches that transformed plants have roots that are at least 8mm long by 16 days and a leaf by at least one month after co-cultivation. The instant specification defines “greenhouse-ready” as a plantlet after it has developed roots that are at least 2cm long and leaves (page 15, lines 10-11). Even if it were the case that roots that were already at least 8mm long would not grow an additional 12mm over the course of 84 days (100 days in the instant claim-16 days in the method of Galan-Avila), it would have been obvious to one of ordinary skill in the art to use a rooting medium as taught by Velcheva and Zhang in order to establish roots on the Cannabis explants. One of ordinary skill in the art would have been motivated to use rooting medium to establish roots because both Velcheva and Zhang rely on rooting medium to generate rooted plants for eventual growing in a greenhouse. One of ordinary skill in the art would have had reasonable expectation of success, because even if one of ordinary skill in the art cultivated the transformed Cannabis shoots on root-induction medium for 5 weeks as taught by Zhang, the total time between inoculation and plants ready for planting in soil would have been 16 days (shoots and roots visible in Galan-Avila) + 35 days (5 weeks rooting induction in Zhang) = 51 days, shorter than the 100 days recited in claim 1. The production of greenhouse-ready plants in less than 100 days would have been obvious over the methods of Velcheva, Galan-Avila, and Zhang.
Thus, claims 1, 3 & 14 are obvious over Velcheva’s method of sterilizing seeds, rinsing seeds in sterile water, excising a hypocotyl explant from the seeds, transforming with a GUS reporter gene via Agrobacterium co-cultivation, culturing on liquid selection medium, and rooting on rooting medium (page 122, left column, paragraph 3-right column, paragraph 3 & page 123, left column, paragraph 2), in view of Galan-Avila. The method is also obvious wherein the seed is a cannabis seed (instant claim 22) and when the exogenous nucleic acid encodes a guide RNA (instant claim 18).
Because the instant specification defines hydration medium as any sterile medium, including sterile water (page 8, lines 1-3) and hydration as a process in which a dry seed takes up water (page 7, lines 25-26), rinsing with sterile distilled water (Velcheva page 122, left column, paragraph 3) reads on hydrating the seed in a hydration medium prior to explant excision (claim 4) in a broad sense.
Galan-Avila’s co-cultivation for 4 days in a medium comprising TDZ reads on claims 6-8, given that TDZ is a growth regulator (instant specification, page 8, lines 12-19). Because the instant specification does not provide a specific definition for force treating, but does provide “addition of chemicals” as an example (page 1), immersing the seeds in NaClO prior to explant removal (Velcheva page 122, left column, paragraph 3) broadly interpreted reads on claims 9 & 10. Galan-Avila’s method achieved transformation rates of greater than 1% in some cultivars, which reads on claim 17.
Claims 1, 3-4, 6-10, 14, 17-18 & 22 are obvious over Velcheva, Galan-Avila, and Zhang.
Applicant urges that the suppression of callus to the degree that callus removal is no longer required in difficult to recover plants and significantly accelerated development of greenhouse-ready plantlets are unexpected results that overcome limitations of transformation of the listed genera of plants that are not inherent or expected, not foreseeable, not inherent, and not predictable (Remarks, page 6, paragraph 2).
This argument is not persuasive, because Galan-Avila teaches a method of cultivating transformed cannabis without callus production, so suppression of callus to the degree that callus removal is no longer required was known in the art for cannabis transformants prior to the effective filing date of the instant application. Likewise, development of plantlets with roots longer than 2 cm would have been obvious over the accelerated development of rooted transgenic explants taught by Galan-Avila. Even if the method of Galan-Avila did not directly result in transformed explants with roots 2cm long by 100 days, and Galan-Avila is silent with respect to this precise phenotypic measurement, methods of rooting cannabis shoots for transplantation into a greenhouse in less than 80 days were known in the art, making the accelerated development recited in claim 1 foreseeable and predictable based on methods known in the art prior to the instant filing.
Because Galan-Avila and Zhang teach that greenhouse ready transformed cannabis explants would have been obvious following methods taught in the art prior to the filing of the instant application, the inherency of greenhouse ready plants or reduced callus to the recited active steps of instant claim 1 is irrelevant to the rejection presented above.
Applicant urges that MPEP 2112 requires a basis in fact and/or technical reasoning to support the determination that the allegedly inherent characteristic necessarily flows from the teachings of the applied prior art. Applicant urges that because the inherency of the recited results was pointed out with reference to the current specification rather than prior art references, the burden of providing a basis in fact or technical reasoning to support the inherency of these elements is not met (Remarks, page 6, paragraphs 3 & page 7, paragraph 3).
This argument is unpersuasive to the rejection above, because Galan-Avila and Zhang teach methods such that greenhouse ready transformed cannabis explants would have been obvious in the art prior to the filing of the instant application. Inherency is not discussed or invoked in the rejection above.
Instant claim 1 does not require any active step beyond excising the explant from a seed by removing a seed coat, introducing an exogenous nucleic acid, and culturing the explant on a liquid selection medium to select for a transformed explant. These active steps were known in the art prior to the filing of the instant application. It would have been obvious to apply the known method to cannabis, as presented in the rejection above.
Applicant urges that because the reducing of callusing and the elimination of the callus-removal step define the specific operational mode and functional outcome of the liquid-selection culture conditions, the wherein clause cannot be treated as aspirational language and is instead functional language that restricts the method to a particular operation configuration and gives meaning and purpose to the recited manipulative steps (Remarks, page 6, paragraph 4-page 7, paragraph 1).
This argument is unpersuasive, because claim 1 does not require that callus is removed or not removed, merely that callus removal is unnecessary. This limitation does not distinguish the claimed method from a method made obvious by the prior art requiring all the recited active steps, especially since methods of transforming and regenerating cannabis without callus removal were known prior to the filing of the instant application. Moreover, methods of transformation and regeneration of cannabis explants that result in plantlets with roots ready for greenhouse transfer in fewer than 100 days were known in and obvious over methods taught in the art prior to the filing of the instant application. The particular operation, configuration, and recited manipulative steps of the instant claims as well as the recited results would have been obvious prior to the filing of the instant application.
Applicant urges that because Velcheva, McCormick and Hesami do not teach or suggest the biological outcomes recited in amended claim 1 including a reduction in callus formation relative to solid-medium methods, those of skill in the art would have no expectation of success in applying the methods to Cannabis, Abelmoschus, Vigna, and Arachis (Remarks, page 7, paragraph 2).
This argument is unpersuasive, because Galan-Avila and Zhang teach and make obvious a method with the recited biological outcomes of reduced callus formation and greenhouse ready plantlets in less than 100 days. One of ordinary skill in the art would have had reasonable expectation of success in achieving the recited outcomes in cannabis by combining the liquid medium culture of Velcheva with the explant culture methods of Galan-Avila and the rooting method of Zhang.
Claim(s) 11 is rejected under 35 U.S.C. 103 as being unpatentable over Velcheva, Galan-Avila, and Zhang as applied to claims 1, 3-4, 6-7, 9-10, 14, 17-18 & 22 above, and further in view of Hesami et al (2021) Int. J. Mol. Sci. 22, 5671 (published 5/26/2021, hereafter Hesami).
This is a new rejection necessitated by Applicant’s amendments filed 3/26/2026.
Claim 11 is drawn to a method wherein the liquid selection medium comprises 10-150 mg/L spectinomycin.
The teachings of Velcheva, Galan-Avila, and Zhang are presented above. They do not teach spectinomycin in the liquid selection medium.
Hesami teaches various media comprising thidizauron (TDZ) for Cannabis culture and teaches that in some genotypes, medium supplemented with 0.1 mg/L TDZ resulted in the highest regeneration frequency (table 2, page 16, row 3). Hesami teaches media for Agrobacterium-mediated gene transformation in Cannabis, including media that comprises 150 mg/L Spectinomycin (table 5, page 30, row 1).
Hesami teaches CRISPR-Cas mediated genome editing in Cannabis as a robust system offering high target specificity (page 37 paragraph 3) that can accelerate the breeding of designer cannabis (page 41, paragraph 1). Hesami teaches that CRISPR/Cas system requires guide RNAs to guide the endonuclease to the specific target DNA (page 37 paragraph 3).
Before the time of filing of the instant application, it would have been obvious to one of ordinary skill in the art to substitute spectinomycin at 150 mg/L for the kanamycin used by Velcheva or Galan-Avila. One of ordinary skill in the art would have been motivated to substitute spectinomycin for kanamycin because Galan-Avila teaches that kanamycin is a poor selectable marker for C. sativa. One of ordinary skill in the art would have had reasonable expectation of success, because spectinomycin had been used as a selectable marker for Cannabis cultures in the prior art.
Claims 1, 3-4, 6-7, 9-11, 14, 17-18 & 22 are obvious over Velcheva, Galan-Avila, Zhang, and Hesami.
Claims 1, 3-12, 14-15, 17-18 & 22 are rejected under 35 U.S.C. 103 being unpatentable over Martinell et al US 8,357,834 B2 (patented 1/22/2013, hereafter Martinell) in view of Petersen et al US 2021/0071186 A1 (published 3/11/2021, over two years prior to the effective filing date of the instant application, hereafter Petersen) and Galan-Avila et al (2021) Industrial Crops & Products. 170. 113691. (available online 6/9/2021, hereafter Galan-Avila) and Zhang et al (2021) Plant Biotechnology Journal. 19: 1979-1987 (published October 2021, hereafter Zhang).
This is a new rejection. Applicant’s remarks filed 3/26/2026 do not address Martinell and Petersen. Applicant’s remarks filed 12/26/2025 with respect to Martinell and Petersen were considered and addressed in the Advisory Action mailed 3/2/2026. They are reconsidered below as applicable to the new rejection but are not persuasive.
Claims 1, 3-4, 6-7, 9-11, 14-15 &-17 are drawn to a method of transforming an explant comprising excising the explant, introducing an exogenous nucleic acid into the plant, and culturing the explant on a liquid selection medium. Claim 8 is drawn to the method wherein the medium comprises dicot INO medium, nystatin, thiabendazole, and thidiazuron. Claim 12 is drawn to the method wherein the liquid selection medium is selected from a group of media. Claim 18 is drawn to the method wherein the exogenous nucleic acid encodes a guide RNA. Claim 22 is drawn to the method wherein the seed is a cannabis seed. Claim 5 is drawn to the method wherein the explant comprises both primary leaves.
Martinell claim 1 teaches a method of transforming an explant from a cotton seed with a heterologous nucleic acid sequence and contacting the explant with a media comprising spectinomycin to select transformed cells (column 53, lines 36-48). Martinell teaches a method of preparing the explant material from soybean seeds by separating the embryo from seed tissues including the seed coat and cotyledons (column 17, line 65-column 18, line 2) or from cotton seeds comprising removing the embryonic axis from the seed, cotyledons, and hypocotyl (column 18, lines 13-17). Martinell teaches that the explant can comprise tissue from the primary leaf (claim 46) but does not require both primary leaves.
Martinell teaches a method where the seed comprising the explant is primed by contacting with an aqueous solution comprising water, specifically cotton seeds were surface sterilized, rinsed, and hydrated in a liquid hydration medium before excision (claim 44; column 18, lines 26-46).
Martinell teaches a method of transforming the cells via Agrobacterium wherein explants are co-cultured with Agrobacterium and exposed to sonication and then incubated for 1-5 days (column 18, line 59-column 19, line14). Martinell also teaches a method of transformation with Agrobacterium wherein inoculation and co-cultivation was carried out for 2-5 days in a medium comprising thidiazuron (TDZ) (column 19, line 47-column 20, line 13).
Martinell teaches a method of transformation wherein after transformation the explants are transferred to liquid medium with a range of spectinomycin (25-250mg/L) (column 22, lines 35-67; claim 49). Martinell teaches that methods involving liquid selection medium with or without spectinomycin had equal or higher transformation frequency than methods comprising selection on semi-solid medium (Table 11 & Table 14).
Martinell teaches a method wherein transformed shoots were rooted on root induction medium and that spectinomycin was added to the medium at ranges of 0-250 ppm or 25mg/L to 250mg/L (column 24, lines 62-column 25, line 12; table 10).
Martinell teaches the time from inoculation to obtaining a plant can be as short as 8 weeks (which is 56 days) (table 11). Martinell teaches that transgenic plants may be regenerated without the need for generating a callus cell culture (column 7, lines 18-22).
Additionally, Martinell teaches that co-culture is performed in INO media comprising glucose (30g/L), MES (3.9g/L), and salts, and brought to a pH of 5.4 with KOH and autoclaved (table 1). Martinell teaches co-culture performed with media comprising TDZ in the range of 0-3 mg/L (table 4). Martinell also teaches the INO medium supplemented with 50 mg/L nystatin and 10 mg/L thiabendazole (TBZ) (column 34, lines 16-21). Martinell teaches that the addition of nystatin (50ppm) and thiabendazole (10ppm) to INO co-culture medium may improve the health of explants (column 9, lines 55-61). Martinell teaches a liquid selection medium comprising 100mg/L timentin to inhibit growth of Agrobacterium and other contaminants (column 20, lines 22-26, table 2).
Martinell does not teach the method in Cannabis, Arachis, Abelmoschus, or Vigna or the co-culture medium comprising dicot INO medium, nystatin, thiabendazole, and thidiazuron, the selection medium from the list in claim 12, or transformation with a nucleic acid encoding a guide RNA.
Petersen teaches an INO inoculation medium for co-culturing Cannabis explants comprising 1.284 g/L Gamborg B5 G398, 30 g/L glucose, 2.8 g/L MES, and adjusted to 5.4 pH with 1N KOH and autoclaved, with 50mg/L nystatin, 10 mg/L TBZ, and 1 mg/L TDZ (table 2, paragraph [0203]).
Additionally, Petersen teaches an agar-based WCIC Hemp Node medium comprising MS salts complete with vitamins (4.43g/L), Cleary’s 336 (0.06 g/L), sucrose (30 mg/L), meta-topolin (0.5mg/L), carbenicillin (250mg/L), and cefotaxime (200 mg/L), autoclaved and adjusted to 5.7 pH with 1N KOH (table 7). Petersen teaches that explants were transferred to this hemp node media comprising 100 mg/L spectinomycin after co-culture (paragraph [0234]). Petersen teaches Gamborg B5 media comprising the selective agent spectinomycin comprising “timetin” (150 mg/L) (table 3 & 6), as well as a hemp meristem regeneration medium comprising 150 mg/L “timetin” (table 9). For purposes of examination, timetin has been interpreted to be the same as timentin in claim 12.
Petersen teaches an example wherein the Cannabis explant is transformed with a nucleic acid encoding a guide RNA targeting a THCA synthase gene, CBDA synthase gene, or EPSP (claim 33, paragraph [0283]). Petersen teaches that tissue culture and regeneration allows the production of clonal and fertile plants (paragraph [0004]). Petersen teaches a motivation to improve Cannabis transformation and gene editing because Cannabis is recalcitrant to produce embryogenic cells for regeneration in culture (paragraph [0004]).
Petersen provides figures of Cannabis transformation wherein embryo explants have cotyledonary tissue removed prior to transformation (Figures 79(2) and 80(2)). Explants with both primary leaves can be seen on the explants where the cotyledonary tissue is removed.
The teachings of Galan-Avila and Zhang are presented above.
Before the time of filing of the instant application, it would have been obvious to one of ordinary skill in the art to substitute the co-culture medium taught by Martinell (table 1) for the co-culture medium taught by Petersen in a method of transformation of explants. One of ordinary skill in the art would have been motivated to make the change based on the rationale of simple substitution of one known element for another to obtain predictable results. One of ordinary skill in the art would have had reasonable expectation of success, because adjusting the concentrations of salts in culture is routine optimization. In addition, it would have been obvious to one of ordinary skill in the art to use 50mg/L nystatin, 10mg/L TBZ, and 1mg/L TDZ in the co-culture medium, because Martinell taught various media for co-culture comprising these values for these components. One of ordinary skill in the art would have been motivated to combine these supplements in the exact ratio presented in instant table 6 through routine optimization. One of ordinary skill in the art would have had reasonable expectation of success, because combinations of media comprising these concentrations of plant growth regulator or antimicrobial substance had been taught by Martinell.
Prior to the effective filing date of the instant application, it would have been obvious to one of ordinary skill in the art to substitute cannabis for cotton and accordingly to substitute the hemp node medium of Petersen (without agar) for the liquid selection medium of Martinell, in order to culture cannabis explants. One of ordinary skill in the art would have been motivated to use the method for cannabis in order to produce clonal plants and to apply the method of Martinell of using spectinomycin for the transformation of Cannabis because kanamycin is a poor selectable marker for cannabis. One of ordinary skill in the art would have been motivated to substitute one medium for another due to the rationale of simple substitution of one known element for another to obtain predictable results. In addition, it would have been obvious to one of ordinary skill in the art to modify the medium to add 150 mg/L timentin as taught by Petersen (tables 3 & 6). One of ordinary skill in the art would have been motivated to add timentin to the medium of Petersen table 7 in order to control the growth of Agrobacterium and other contaminants. One of ordinary skill would have had reasonable expectation of success, because various concentrations of timentin are used in media taught by Petersen.
Before the time of filing of the instant application, it would have been obvious to one of ordinary skill in the art to use an exogenous nucleic acid encoding a guide RNA for transformation of the explant, as taught by Petersen. One of ordinary skill in the art would have been motivated to use a CRISPR/Cas system with a guide RNA because gene editing was taught to be a new approach for Cannabis breeding and a successful method of transforming cannabis with a guide RNA had been taught by Zhang. One of ordinary skill in the art would have had reasonable expectation of success in light of Zhang’s success.
It would have been obvious to use an explant comprising both primary leaves, since explants with both primary leaves were used in the method of Petersen (instant claim 5).
Galan-Avila does not explicitly teach that the rooted Cannabis explants are greenhouse-ready in fewer than 100 days after introduction of the exogenous nucleic acid. Galan-Avila teaches that transformed plants have roots that are at least 8mm long by 16 days and a leaf by at least one month after co-cultivation. The instant specification defines “greenhouse-ready” as a plantlet after it has developed roots that are at least 2cm long and leaves (page 15, lines 10-11). Even if it were the case that roots that were already at least 8mm long would not grow an additional 12mm over the course of 84 days (100 days in the instant claim-16 days in the method of Galan-Avila), it would have been obvious to one of ordinary skill in the art to use a rooting medium as taught by Zhang in order to establish roots on the Cannabis explants. One of ordinary skill in the art would have been motivated to use rooting medium to establish roots because Zhang relies on rooting medium to generate rooted plants for eventual growing in a greenhouse. One of ordinary skill in the art would have had reasonable expectation of success, because even if one of ordinary skill in the art cultivated the transformed Cannabis shoots on root-induction medium for 5 weeks as taught by Zhang, the total time between inoculation and plants ready for planting in soil would have been 16 days (shoots and roots visible in Galan-Avila) + 35 days (5 weeks rooting induction in Zhang) = 51 days, shorter than the 100 days recited in claim 1.
Finally, Galan-Avila’s method required no callus removal step, which reads on callusing reduced to the degree that callus no longer needs to be removed from the explants. It is noted that although instant claim 1 recites that callus no longer needs to be removed from the explants, the method of claim 1 does not require that the callus is removed or is not removed.
Thus, claims 1, 3-12, 14-15, 17-18 & 22 are unpatentable over Martinell in view of Petersen, Galan-Avila and Zhang.
Applicant urges that Martinell and Petersen do not teach that liquid culture would mitigate the unorganized callusing of cannabis or a reduction in callus. Applicant urges that this result is not inherent to the method and urges that inherency of the recited result is not met (remarks mailed 12/26/2025, page 8, paragraph 2).
This argument is unpersuasive, because claim 1 does not require that callus is removed or not removed, merely that callus removal is unnecessary. This limitation does not distinguish the claimed method from a method made obvious by the prior art requiring all the recited active steps, especially since methods of transforming and regenerating cannabis without callus removal were known prior to the filing of the instant application. Methods of transformation and regeneration of cannabis explants that result in plantlets with roots ready for greenhouse transfer in fewer than 100 days were also known in and obvious over methods taught in the art prior to the filing of the instant application.
Claim(s) 1, 3, 4, 6-7, 9, 10, 12, 14, 17 & 23 are rejected under 35 U.S.C. 103 as being unpatentable over Velcheva in view of Raveendar et al (2010) Asian Journal of Plant Sciences. 9(5): 256-263. (published 2010, hereafter Raveendar).
Claim 23 is drawn to the method wherein the seed is from Vigna unguiculata.
The teachings of Velcheva are presented above. Velcheva does not teach a method wherein the seed is Abelmoschus, Vigna, or Arachis seed or the length of time that Agrobacterium are co-cultivated with explants, nor the medium in which co-culture is performed. Velcheva does not teach a liquid selection medium from the list in claim 12.
Raveendar teaches a meristem-targeted rapid and efficient transformation system in cowpea (page 256, right column, paragraph 2). Cotyledonary node explants were excised from in vitro seedlings by surface sterilizing seeds with HgCl2, washing seeds in sterile distilled water, culturing on MSB5-G medium, and removing cotyledons and cutting epicotyls and hypocotyls (page 257, left column, paragraph 2). Explants were transformed with A. tumefaciens on MSB5-CCM coculture medium for 3 days, cultured on selection medium, transferred onto shoot elongation medium and cultured for a total of 4-6 weeks, and rooted on MSB basal medium before establishing in soil and transferring to the greenhouse (page 257, right column, paragraph 2-page 258, left column, paragraph 1). Raveendar teaches that regenerated transformants can be obtained within 7 weeks (which is 49 days) after Agrobacterium inoculation (page 260, right column, paragraph 2; figure 5). A plant with roots in root induction media is shown in figure 2c, although no scale bar is provided.
MSB5-CCM coculture medium comprises MS salts and 6.6 uM BAP and the MSB5 selection medium comprised MS salts (table 1).
Transformation efficiency of cotyledonary node explants of Vigna unguiculata was greater than 1% (table 3).
Raveendar teaches that Vigna unguiculata is an important food legume, and genetic improvement will be useful for development of increased quality and yield; however, routine genetic transformation protocols are limited due to inability to regenerate under tissue culture conditions (page 256, left column, paragraph 1).
Before the time of filing of the instant application, one of ordinary skill in the art would have been motivated to modify the method of Velcheva to apply the liquid culture medium to Vigna unguiculata transformation. One of ordinary skill in the art would have been motivated to apply Velcheva’s method to limit escapes and improve transformation to Vigna unguiculata because Vigna unguiculata has limited transformation protocols despite being an important food legume. One of ordinary skill in the art would have had reasonable expectation of success, because Vigna unguiculata was capable of regenerating after Agrobacterium-mediated transformation following other protocols.
The instant specification defines “greenhouse-ready” as a plantlet after it has developed roots that are at least 2cm long and leaves (page 15, lines 10-11). Raveendar is silent with regard to the length of the roots of the plants that were transferred to the greenhouse in 7 weeks. The root shown on the plant on rooting medium is roughly as long as the diameter of the test tube it is grown in. At least some of the plants that would be transplanted in soil in Raveendar’s 49 days would be expected to have roots 2 cm long in the next 50 days, which reads on plantlets that are greenhouse ready according to the instant definition produced in fewer than 100 days.
Although instant claim 1 recites that callus no longer needs to be removed from the explants, the method of claim 1 does not require that the callus is removed or is not removed. Raveendar’s method does not teach a callus removal step, which reads on callusing reduced to the degree that callus no longer needs to be removed from the explants.
Because the instant specification defines hydration medium as any sterile medium, including sterile water (page 8, lines 1-3) and hydration as a process in which a dry seed takes up water (page 7, lines 25-26), rinsing with sterile distilled water reads on hydrating the seed in a hydration medium prior to explant excision (claim 4) in a broad sense.
Raveendar’s co-cultivation for 3 days in a medium comprising BAP reads on claims 6-7, given that BAP is a growth regulator (instant specification, page 8, lines 12-19). Because the instant specification does not provide a specific definition for force treating, but does provide “addition of chemicals” as an example (page 1), immersing the seeds in NaClO prior to explant removal (Velcheva page 122, left column, paragraph 3) broadly interpreted reads on claims 9 & 10. Raveendar’s method achieved transformation rates of greater than 1% in some cultivars, which reads on claim 17. Raveendar’s selection medium comprising MS salts reads on a MS salts-based medium. It would have been obvious to modify the liquid selection medium of Velcheva to use MS salts-based medium such as Raveendar’s to optimize for the culture of Vigna. One of ordinary skill in the art would have had reasonable expectation of success because optimization of medium was routine prior to the instant filing, and Raveendar teaches that MS-salts based medium was appropriate for Vigna, making instant claim 12 obvious.
Thus, Velchova, in light of Raveendar, makes obvious claims 1, 3, 4, 6-7, 9, 10, 12, 14, 17 & 23.
Conclusion
No claims are allowed.
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/VICTORIA L DELEO/Examiner, Art Unit 1662
/Anne Kubelik/Primary Examiner, Art Unit 1663
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