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 .
Status of the Claims
Claims 2-3 and 6 are canceled.
Claims 1, 4-5, and 7-10 are pending.
Claims 1, 4-5, and 7-10 are examined herein.
The objections to claims 1-10 have been withdrawn in view of Applicant’s amendments to the claims and/or Applicant’s arguments.
The rejections to claims 1-10 under 35 USC § 112(b) have been withdrawn in view of Applicant’s amendments to the claims.
Claims 1, 4-5, and 7-10 are examined rejected.
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. Application No. 18/652,906 filed on 05/02/2024 claims foreign priority to Chinese Application No. CN202310994490.0 filed on 08/09/2023.
A certified English translation has been filed and is labeled as a Specification document dated 10/09/2025, and a Transmittal Letter document dated 10/09/2025.
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 1 is rejected under 35 U.S.C. 103 as being unpatentable over Xiang (Xiang, Z., Chen, Y., Chen, Y., Zhang, L., Liu, M., Mao, D., & Chen, L. (2022). Agrobacterium-mediated high-efficiency genetic transformation and genome editing of chaling common wild rice (Oryza rufipogon griff.) using scutellum tissue of embryos in mature seeds. Frontiers in Plant Science, 13, 849666), Jyothishwaran (Jyothishwaran, G., Kotresha, D., Selvaraj, T., Srideshikan, S. M., Rajvanshi, P. K., & Jayabaskaran, C. (2007). A modified freeze-thaw method for efficient transformation of Agrobacterium tumefaciens. Current science, 93(6), 770-772), Vain (Vain, P., Worland, B., Kohli, A., Snape, J. W., & Christou, P. (1998). The green fluorescent protein (GFP) as a vital screenable marker in rice transformation. Theoretical and Applied Genetics, 96, 164- 169), Yaqoob (Yagoob. U., Kaul, T., & Nawchoo, I. A. (2017). Development of an efficient protocol for Agrobacterium mediated transformation of some recalcitrant indica rice varieties. Indian Journal of Plant Physiology, 22, 346-353), Ozawa (Ozawa, K. (2009). Establishment of a high efficiency Agrobacterium-mediated transformation system of rice (Oryza sativa L.). Plant Science, 176(4), 522- 527), Wang (Wang, M., Yan, F., & Zhou, H. (2022). Protocol for targeted modification of the rice genome using base editing. STAR protocols, 3(4), 101865), and Wanichananan (Wanichananan, P., Teerakathiti, T., Roytrakul, S., Kirdmanee, C., & Peyachoknagul, S. (2010). A highly efficient method for Agrobacterium mediated transformation in elite rice varieties (Oryza sativa L. spp. indica). African Journal of Biotechnology, %34)), and Guo (Guo, H. B., Li, S. M., Peng, J., & Ke, W. D. (2007). Zizania latifolia Turcz. cultivated in China. Genetic Resources and Crop Evolution, 54(6), 1211-1217).
This is a modified rejection from the previous rejection set forth in the Office Action dated 07/10/2025, necessitated by Applicant’s amendments.
Claim 1 is drawn to a method for gene expression by transient transformation of a Zizania latifolia seed using Agrobacterium, comprising the following steps:(1) inoculating a sterilized Zizania latifolia seed into an induction medium to allow induction culture of a callus, conducting subculture, and selecting an embryonic callus to allow pre-culture to obtain a pre-cultured callus;(2) transforming a plasmid carrying a green fluorescent protein (GFP) gene into an Agrobacterium strain EHA105 or LBA4404 to allow culture at 28°C for 2 days (d). selecting a resulting single clone to allow PCR and electrophoresis detection to obtain a positive Agrobacterium strain; transferring the positive Agrobacterium strain into a yeast extract broth (YEB) liquid medium to allow culture, subjecting an obtained bacterial solution to centrifugation to collect a bacterial cell, resuspending the bacterial cell in an infection buffer until a resulting bacterial suspension has an optical density (OD) value of 0.02 to 0.5 at a wavelength of 600 nm to obtain an infection bacterial solution; and(3) infecting the pre-cultured callus with the infection bacterial solution, inoculating a resulting infected callus into a co-culture medium to allow co-culture in the dark at 22°C±2°C for 2 d to 4d; transferring a resulting co-cultured callus into a recovery medium to allow recovery culture in the dark at 28°C±2°C for 3 d; observing an infection status of a recovered callus under a stereo fluorescence microscope, wherein the GFP gene serving as an exogenous target gene is successfully expressed in the callus of the Zizania latifolia seed if the recovered callus has green fluorescence under ultraviolet light; and calculating a transient expression rate; wherein the Agrobacterium strain is selected from the group consisting of Agrobacterium strains EHA105 and LBA4404,wherein the co-culture is conducted for 2-3 d, wherein the bacterial suspension has an OD value of 0.2 at a wavelength of 600 nm; and wherein 50 mg/mL kanamycin and 50 mg/mL rifampicin are added into the YEB liquid medium, and the centrifugation is conducted at 1,000 rpm to 2,000 rpm for 5 minutes (min) in step (2).
Regarding claim 1, Xiang teaches sterilized wild rice seeds were cultured on an induction medium to induce callus and subsequently sub-cultured, and callus was selected to be transformed (p. 3, section titled Tissue Culture and Agrobacterium-Mediated Transformation, Fig. 3B and 3C). Xiang also teaches transforming a reporter vector into Agrobacterium tumefaciens strain EHA105 and culturing it at 28°C for 2-3 days on selective media to select a single colony carrying the plasmid, culturing the single colony in YEB liquid media, and collecting and diluting the bacteria with a bacterial infection suspension to an OD600 of approximately 0.1 for callus infection (p. 3, section titled Tissue Culture and Agrobacterium-Mediated Transformation). Furthermore, Xiang teaches immersing the sub-cultured callus in the bacterial suspension (i.e. infecting the pre-cultured callus with the infection bacterial solution) and placing the infected calli on a co-cultivation medium for 3 days at 25°C (p. 3, Col. 2) (i.e. 22 ± 2°C encompasses 24 ±2C which is merely close to 25 °C taught by Xiang, therefore the claimed range is obvious in view of Xiang; and wherein co-culture is conducted for 2-3 days). Then, Xiang teaches transferring the calli to a selection medium for culture at 28 °C in the dark (p. 3, Col. 2), which is reasonably interpreted as a recovery medium to allow recovery culture because the purpose of the selection medium is to recover transgenic calli. Xiang further teaches calculating transformation efficiency (p. 4, section titled Calculation Formulae, Fig. 3 H-J).
However, Xiang does not explicitly teach the following limitations:
The rice species is Zizania latifolia
The plasmid is carrying a GFP gene
The resulting single colony that was selected in part 2 was confirmed positive via PCR/ gel electrophoresis
the co-culture step occurred in the dark
observing an infection status of a recovered callus under a stereo fluorescence microscope, wherein the GFP gene serving as an exogenous target gene is successfully expressed in the callus of the Zizania latifolia seed if the recovered callus appears green fluorescence under ultraviolet light
wherein the bacterial suspension has an OD value of 0.2 at a wavelength of 600 nm
wherein 50 mg/mL kanamycin and 50 mg/mL rifampicin are added into the YEB liquid medium, and the centrifugation is conducted at 1,000 rpm to 2,000 rpm for 5 minutes (min) in step (2).
In analogous art, Guo teaches Zizania latifolia is a known rice cultivar that has been cultivated for more than 2000 years (abstract), and has high nutritional value (p. 1212, ¶1). In other analogous art, Jyothishwaran teaches a method of introducing plasmids into Agrobacterium tumefaciens, and specifically teaches confirming the Agrobacterium transformants are positive for the plasmid vector using PCR and gel electrophoresis (p. 771, Col. 3). In analogous art related to transformation of embryogenic rice calli, Vain teaches transformation of rice callus using a plasmid comprising a GFP gene (abstract, p. 165, Materials and Methods section titled Rice transformation), and the plasmid resulted in constitutive expression of GFP and allowed for easily detectable and screenable bright-green fluorescence in rice tissue (abstract). Vain also teaches using an ultraviolet lamp to identify recovered GFP-expressing callus, and detection of GFP was monitored at a higher magnification using a Nikon Microphot-5A fluorescent microscope (i.e. a stereo fluorescence microscope) (p. 165, sections titled Visual selection of GFP-fluorescent tissue in vitro, and Detection of GFP expression by fluorescence microscopy). In other analogous art related to the development of an efficient protocol for Agrobacterium-mediated transformation of some recalcitrant rice varieties, Yaqoob teaches the co-cultivation step occurs in the dark for 2 days at 22- 28°C (i.e. 25 °C), then the calli is cultured on resting media in the dark at 25 °C (p. 347, section titled Agrobacterium tumefaciens strain and transformation) which is reasonably interpreted as merely close to the claimed temperature of 28 °C. In analogous art related to the establishment of a high efficiency Agrobacterium-mediated transformation system of rice (title), Ozawa teaches the best conditions for cocultivation of rice calli are at an Agrobacterium concentration of OD = 0.2 (p. 525, Col. 2, and Table 2). Furthermore, in other analogous art, Wang teaches the Agrobacterium can also be cultured in medium containing 50 mg/L Kanamycin and 50 mg/L Rifampicin (p. 11, step 17a), and teaches the stock solutions of kanamycin and rifampicin were both 50 mg/mL (p. 4). In other analogous art, Wanichananan teaches the centrifugation step occurred at 500 rpm over a period of 5 minutes (p. 5489, section titled Optimization of Agrobacterium mediated transformation methods).
It would therefore have been obvious to a person of ordinary skill in the art to modify the invention of Xiang to include the limitations of Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo to arrive at the instantly claimed method with a reasonable expectation of success because the additional limitations are routine steps known in the art and can be achieved without encountering any special technical difficulties. One having ordinary skill in the art would have been motivated to combine the method of Xiang with the limitation of Jyothishwaran because the additional step of confirming the Agrobacterium colony is positive for the plasmid using PCR and gel electrophoresis is a quick and simple method to confirm the bacteria is carrying the plasmid (p. 771, Col. 3). One having ordinary skill in the art would have been motivated to combine the method of Xiang with the limitations of Vain because Vain teaches using the GFP gene as a screenable marker in rice transformation allows for easily screenable and detectable transformed rice tissue (abstract). One having ordinary skill in the art would have been motivated to combine the method of Xiang with the limitations of Yagoob because Yaqoob teaches transformation methods of rice, including recalcitrant varieties, and explicitly teaches necessary conditions including length of co-culture and resting periods, culturing the callus in dark conditions, and temperature requirements to culture and obtain high transformation efficiency of rice callus (Title, abstract, Fig. 2). Additionally, Ozawa teaches an OD600 of 0.2 results in the best transformation efficiency, and one of ordinary skill could incorporate the specified parameter by diluting the bacterial culture to an OD600 of 0.2 taught by Ozawa instead of 0.1 taught by Xiang without encountering any special technical obstacles . One having ordinary skill in the art would have been motivated to do so because Ozawa teaches an OD600 of 0.2 results in the best transformation efficiency of rice (p. 525, Col. 2, and Table 2). The methods of Wang also resulted in Agrobacterium growth harboring the plasmid, and the teachings of Wang and Wanichananan both result in the pelleted Agrobacterium that is to be used for plant transformation. One of ordinary skill could incorporate the method of centrifugation at 2000 rpm and for 5 minutes without encountering any special technical obstacles. One having ordinary skill in the art would have been motivated to combine the teachings because Wang and Wanichananan teach successful growing and pelleting of the Agrobacterium, and it would be prima facie obvious to centrifuge at any speed that collects viable Agrobacterium for transformation including at a low speed of 2000 rpm to prevent bacterial cell death, and to centrifuge at 5 minutes to complete the transformation protocol in a shorter period of time. Furthermore, one having ordinary skill in the art would have been motivated to combine the teachings with Guo because Guo teaches Zizania latifolia is a known rice cultivar with nutritional benefits and is also susceptible to disease (p. 1211-1212), and it would be prima facie obvious to apply known transformation methods to another known cultivar for the same purpose of Agrobacterium-mediated transformation. It would further be obvious to combine the teachings and select Zizania latifolia for transformation for the purpose of aiming to enhance its resistance to a known disease it is susceptible to.
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo as applied to claim 1 above, and further in view of Cho (Cho, M. J., Yano, H., Okamoto, D., Kim, H. K., Jung, H. R., Newcomb, K., ... & Lemaux, P. G. (2004). Stable transformation of rice (Oryza sativa L.) via microprojectile bombardment of highly regenerative, green tissues derived from mature seed. Plant cell reports, 22, 483-489), Sasaki (Sasaki, K., Kishitani, S., Abe, F., & Sato, T. (2005). Promotion of seedling growth of seeds of rice (Oryza sativa L. cv. Hitomebore) by treatment with H202 before sowing. Plant Production Science, 8(5), 509-514) and Shri (Shri, M., Rai, A., Verma, P. K., Misra, P., Dubey, S., Kumar, S., ... & Chakrabarty, D. (2013). An improved Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.) cultivars. Protoplasma, 250(2), 631-636).
This is a modified rejection from the previous rejection set forth in the Office Action dated 07/10/2025, necessitated by Applicant’s amendments.
Claim 4 is drawn to the method according to claim 1, wherein a preparation process of the sterilized Zizania latifolia seed in step (1) comprises: immersing a Zizania latifolia seed in 75% ethanol for 30 seconds (s) to 60 s and in 20% sodium hypochlorite for 20 min, washing a resulting immersed Zizania latifolia seed with sterile water 5 to 7 times until an obtained washing solution is clear, and then immersing a resulting washed Zizania latifolia seed in the sterile water overnight; and an embryo of the sterilized Zizania latifolia seed is inoculated into the induction medium to allow germination, and the induction culture of the callus is conducted at 28°C±2°C for 4 to 5 weeks in the dark to obtain an embryogenic callus; an embryogenic callus with a light yellow appearance is selected to allow the subculture on the induction medium in the dark at 28°C±2°C; a resulting subcultured embryogenic callus is selected to allow the pre-culture in the dark at 28°C±2°C for 7 d to 9 d.
Regarding claim 4, Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo teach the limitations of claim 1 as set forth in the previous obviousness rejection. The teachings of Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo as they are applied to claim 1 are set forth previously herein and are incorporated by reference. Furthermore Xiang teaches surface sterilizing the wild rice seed in 75% EtOH for 3 minutes (i.e. because Xiang teaches sterilizing the seed in 75% EtOH for 3 minutes, the seeds must have been sterilized for 30 to 60 seconds), then 3% sodium hypochlorite for 40 minutes (i.e. because Xiang teaches immersing the seed in sodium hypochlorite for 40 minutes, the seeds must have been immersed in sodium hypochlorite for 20 minutes), washing the seeds with sterile water 5 times, transferring the seeds to an induction medium for 35 days (i.e. 5 weeks) at 28°C (p.3, Col. 1). Additionally, Xiang teaches selecting the embryogenic callus that has a light yellow appearance is selected to allow the subculture on the induction medium (p. 3, Col. 1, Fig. 3A-C).
However, Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo do not explicitly teach:
the sodium hypochlorite solution is 20%
the seed is immersed in sterile water overnight
callus induction and sub-culture occurring in dark conditions iv. the resulting sub-culture is selected to allow the preculture in the dark at for 7 to 9 days
In analogous art, Cho teaches surface sterilizing rice seeds with 20% bleach for 20 minutes (p. 484, section titled Plant material and culture of explants). In other analogous art, Shri teaches callus is incubated in the dark (i.e. reasonably interpreted as both callus induction and sub-culturing occurs in the dark) (p. 632, Col. 1). Shri also teaches the embryogenic calli were sub-cultured onto fresh medium 10 days before transformation (p. 632, Col. 1). In other analogous, Sasaki teaches after sterilization, rice seeds are imbibed in sterile water for 24 hours (p. 10, section titled plant material and growth).
It would therefore have been obvious to a person of ordinary skill in the art to modify the invention of Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo to include the limitations of Cho, Shri, and Sasaki to arrive at the instantly claimed method with a reasonable expectation of success because Cho, Shri, and Sasaki teach rice callus induction and transformation methods, and incorporating the specific limitations could be achieved by a person of ordinary skill in the art without encountering any special technical obstacles. One having ordinary skill in the art would have been motivated to do so because Cho teaches an increased sodium hypochlorite concentration to effectively sterilize the rice seeds for tissue culture (p. 484, section titled Plant material and culture of explants), Shri teaches an improved Agrobacterium-mediated transformation or recalcitrant rice cultivars (title), and Sasaki teaches a method of promoting seedling growth of rice seeds (title), and imbibition is also known in the art to improve germination rates of rice. Furthermore, it would be prima facie obvious to allow pre-culture in the dark for 7-9 days because Shri teaches pre-culture for 10 days, and a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close (See MPEP 2144.05 I).
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo as applied to claim 1 above, and further in view of Zaidi (Zaidi, M. A., Narayanan, M., Sardana, R., Taga, |., Postel, S., Johns, R., ... & Altosaar, I. (2006). Optimizing tissue culture media for efficient transformation of different indica rice genotypes. Agron Res, 4(2), 563-575) and Mostafiz (Binte Mostafiz, S.. & Wagiran, A. (2018). Efficient callus induction and regeneration in selected indica rice. Agronomy, 8(5), 77).
This is a modified rejection from the previous rejection set forth in the Office Action dated 07/10/2025, necessitated by Applicant’s amendments.
Claim 5 is drawn to The method according to claim 1, wherein the induction medium in step (1) comprises: 4.43 grams (g) of Murashige & Skoog basal salt mixture (MS salt), 1 mL of 2 mg/mL 2,4-D, 0.5 g of casein hydrolysate, 30 g of sucrose, 0.1 g of inositol, and 3 g of phytagel per liter of the induction medium with a pH value of 5.7.
Regarding claim 5, Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo teach the limitations of claim 1 as set forth in the previous obviousness rejection. The teachings of Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo as they are applied to claim 1 are set forth previously herein and are incorporated by reference. Furthermore, Xiang teaches the callus induction media comprised 2.5 mg of 2,4-D, 0.6 g of casein hydrolysate, 30 g of sucrose, 0.1 g of inositol, 4.6 g of phytagel, and was pH'd to 5.8.
However, Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo do not explicitly teach the medium was MS-based, the claimed concentrations of 2,4-D, casein hydrolysate, phytagel, and the pH is 5.7.
In analogous art related to improving callus induction media, Mostafiz teaches adjusting the callus induction medium to a pH of 5.7 (p. 3, section titled 2.3 Callus Induction Experiment). In other analogous art related to optimizing tissue culture media for efficient transformation of different rice genotypes, Zaidi teaches the optimized callus induction media consisted of basic MS salt mixtures (abstract). Zaidi further teaches evaluating various concentrations and types of basic media, carbohydrate sources and concentrations, agar concentrations, amino acids, cytokinins and auxins is important to optimize callus induction of various rice genotypes (abstract).
It would therefore have been obvious to a person of ordinary skill in the art to modify the invention of Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo to include the limitations of Mostafiz and Zaidi to arrive at the instantly claimed method with a reasonable expectation of success because Mostafiz and Zaidi teach adjustments to media components is necessary to determine an optimal callus induction medium, and one of ordinary skill in the art could make concentration and pH adjustments without encountering any special technical obstacles. One having ordinary skill in the art would have been motivated to do so because Zaidi teaches various adjustments to concentrations can improve callus induction in different rice genotypes (abstract), and it would therefore be prima facie obvious to adjust concentrations of media components to arrive at the instantly claimed method.
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo as applied to claim 1 above, and further in view of Thao (Thao, B. P., Linh, N. T., Van Manh, N., Linh, L. K., Ha, C. H., Phat, D. T., & Ngoc, P. B. (2022). Optimization of Agrobacterium-mediated transformation procedure for an indica rice variety-Khang Dan 18. Vietnam Journal of Biotechnology, 20(1), 53- 62) and Zaidi (Zaidi, M. A., Narayanan, M., Sardana, R., Taga, l., Postel, S., Johns, R., ... & Altosaar, |. (2006). Optimizing tissue culture media for efficient transformation of different indica rice genotypes. Agron Res, 4(2), 563-575).
This is a modified rejection from the previous rejection set forth in the Office Action dated 07/10/2025, necessitated by Applicant’s amendments.
Claim 7 is drawn to the method according to claim 1, wherein each liter of the infection buffer in step (2) comprises 4 g of trace element-containing CHU' S N6 BA SAL SALT MIX (N6) 1 mL of 200 µM/mL acetosyringone, 200 µL of 10 mg/mL 2,4-D, 1 g of casein hydrolysate, 30 g of sucrose, 0.1 g of inositol, and 10 g of glucose.
Regarding claim 7, Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo teach the limitations of claim 1 as set forth in the previous obviousness rejection. The teachings of Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo as they are applied to claim 1 are set forth previously herein and are incorporated by reference. Furthermore, Xiang teaches the infection buffer (i.e. AA suspension buffer) comprises 20 mg of acetosyringone, 500 mg casein hydrolysate, 68.6 g of sucrose, 100 mg of inositol, and 36 g of glucose (Supplementary Table 2).
However, Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo do not explicitly teach the medium is N6-based medium, the medium comprises 2,4-D, nor do they explicitly teach the specified concentrations of the media components. In analogous art, Thao teaches suspending the Agrobacterium cells in N6 liquid medium for transformation (p. 55, section titled Agrobacterium suspension preparation). In other analogous art, Wang teaches resuspending Agrobacterium in liquid medium comprising 2,4-D (p.5, p. 11, step 17c). In other analogous art related to optimizing tissue culture media for efficient transformation of different rice genotypes, Zaidi teaches the importance of optimizing media by adjusting various concentrations and types of basic media, carbohydrate sources and concentrations, agar concentrations, amino acids, cytokinins and auxins is important to optimize callus induction and transformation of various rice genotypes (abstract).
It would therefore have been obvious to a person of ordinary skill in the art to modify the invention of as taught by Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo to include the limitations of Thao and Zaidi to arrive at the instantly claimed method with a reasonable expectation of success because Thao and Zaidi teach media necessary for Agrobacterium-mediated transformation, and Thao teaches an alternative base media and Zaidi teaches the importance of making adjustments to concentrations of the media components to optimize transformation efficiency. One of ordinary skill in the art could incorporate the N6 basic media and make concentration adjustments without encountering any special technical obstacles. One having ordinary skill in the art would have been motivated to do so because Zaidi teaches various adjustments to concentrations can improve transformation efficiency in different rice genotypes (title, abstract), and it would therefore be prima facie obvious to adjust concentrations of media components to arrive at the instantly claimed method.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo as applied to claim 1 above, and further in view of Thao (Thao, B. P., Linh, N. T., Van Manh, N., Linh, L. K., Ha, C. H., Phat, D. T., & Ngoc, P. B. (2022). Optimization of Agrobacterium-mediated transformation procedure for an indica rice variety-Khang Dan 18. Vietnam Journal of Biotechnology, 20(1), 53- 62).
This is a modified rejection from the previous rejection set forth in the Office Action dated 07/10/2025, necessitated by Applicant’s amendments.
Claim 8 is drawn to the method according to claim 1, wherein the pre-cultured callus is infected with the infection bacterial solution for 20 min, and the infected callus is co-cultured on the co-culture medium for 3 d in step (3).
Regarding claim 8, Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo teach the limitations of claim 1 as set forth in the previous obviousness rejection. The teachings of Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo as they are applied to claim 1 are set forth previously herein and are incorporated by reference. Furthermore, as previously described in the rejection of claim 1, Xiang teaches co-cultivating the infected calli on a co-culture medium for 3 days (p. 3, Col. 2).
However, Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo do not explicitly teach wherein the pre-cultured callus is infected with the infection bacterial solution for 20 min.
In analogous art, Thao teaches the calli were infected with the Agrobacterium culture for 10, 20, and 30 minutes, all of which successfully produced transient and stable transgene expression in transgenic callus and 20 minutes of infection was optimal (Table 5).
It would therefore have been obvious to a person of ordinary skill in the art to modify the invention of Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo to include the limitations of Thao to arrive at the instantly claimed method with a reasonable expectation of success because the method of Thao teaches 20 minutes of bacterial infection was optimal, and one of ordinary skill could infect the callus for 20 minutes as taught by Thao without encountering any special technical obstacles. One having ordinary skill in the art would have been motivated to do so because Thao teaches a transformation method with optimized parameters, and specifically teaches the optimal infection time being 20 minutes (Table 5). It would have been prima facie obvious to apply the optimized parameter of a 20 minute infection period of one rice variety to another rice variety for the same purpose.
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo as applied to claim 1 above, and further in view of Mostafiz_(Binte Mostafiz, S., & Wagiran, A. (2018). Efficient callus induction and regeneration in selected indica rice. Agronomy, 8(5), 77) and Zaidi (Zaidi, M. A., Narayanan, M., Sardana, R., Taga, |., Postel, S., Johns, R., ... & Altosaar, |. (2006). Optimizing tissue culture media for efficient transformation of different indica rice genotypes. Agron Res, 4(2), 563-575).
This is a modified rejection from the previous rejection set forth in the Office Action dated 07/10/2025, necessitated by Applicant’s amendments.
Claim 9 is drawn to the method according to claim 1, wherein each liter of the infection buffer in step (2) comprises 4 g of trace element-containing CHU’S N6 BA SAL SALT MIX (N6) 1 mL of 200 µM/mL acetosyringone, 200 µL of 10 mg/mL 2,4-D, 1 g of casein hydrolysate, 30 g of sucrose, 0.1 g of inositol, and 10 g of glucose.
Regarding claim 9, Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo teach the limitations of claim 1 as set forth in the previous obviousness rejection. The teachings of Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo as they are applied to claim 1 are set forth previously herein and are incorporated by reference. Furthermore, Xiang teaches the co-cultivation medium comprises N6 macronutrient, 20 mg of acetosyringone, 2.5 mg of 2,4-D, 600 mg casein hydrolysate, 30 g of sucrose, 100 mg of inositol, and 10 g of glucose, 4.6 g of phytagel, and pH is 5.8 (Supplementary Table 2).
However, Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo do not explicitly teach the specified concentrations of the media components, nor do they teach the pH is 5.7.
In analogous art related to improving media for rice callus formation, Mostafiz teaches adjusting the medium to a pH of 5.7 (p. 3, section titled 2.3 Callus Induction Experiment). In other analogous art related to optimizing tissue culture media for efficient transformation of different rice genotypes, Zaidi teaches the importance of optimizing media by adjusting various concentrations and types of basic media, carbohydrate sources and concentrations, agar concentrations, amino acids, cytokinins and auxins is important to optimize callus induction and transformation of various rice genotypes (abstract).
It would therefore have been obvious to a person of ordinary skill in the art to modify the invention of as taught by Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo to include the limitations of Mostafiz and Zaidi to arrive at the instantly claimed method with a reasonable expectation of success because Mostafiz and Zaidi teach media necessary for callus growth and Agrobacterium-mediated transformation. Specifically, Mostafiz teaches successful callus growth on a media with a pH of 5.7 (p. 3), and it is known in the art that it important to promote callus growth following transformation. Furthermore, Zaidi teaches the importance of making adjustments to concentrations of the media components to optimize transformation efficiency. One of ordinary skill in the art could incorporate the pH of 5.7 and make concentration adjustments without encountering any special technical obstacles. One having ordinary skill in the art would have been motivated to do so because Zaidi teaches various adjustments to concentrations can improve transformation efficiency in different rice genotypes (title, abstract), and it would therefore be prima facie obvious to adjust concentrations of media components and adjust the pH to a value for optimal nutrient uptake and callus growth to arrive at the instantly claimed method.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo as applied to claim 1 above, and further in view of Wang (Wang, M., Yan, F., & Zhou, H. (2022). Protocol for targeted modification of the rice genome using base editing. STAR protocols, 3(4), 101865), Mostafiz (Binte Mostafiz, S., & Wagiran, A. (2018). Efficient callus induction and regeneration in selected indica rice. Agronomy, 8(5), 77) and Zaidi (Zaidi, M. A., Narayanan, M. Sardana, R., Taga, |., Postel, S., Johns, R., ... & Altosaar, I. (2006). Optimizing tissue culture media for efficient transformation of different indica rice genotypes. Agron Res, 4(2), 563-575).
This is a modified rejection from the previous rejection set forth in the Office Action dated 07/10/2025, necessitated by Applicant’s amendments.
Claim 10 is drawn to the method according to claim 1, wherein the recovery medium in step (3) comprises: 4.43 g of MS salt, 0.5 g of casein hydrolysate, 2 mL of 2 mg/mL 2,4-D, 100 µL of 1 mg/mL 6-BA, 2 mL of 200 mg/mL Timentin, 0.1 g of inositol, 30 g of sucrose, and 3 g of phytagel per liter of the recovery medium with a pH value of 5.7.
Regarding claim 10, Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo teach the limitations of claim 1 as set forth in the previous obviousness rejection. The teachings of Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo as they are applied to claim 1 are set forth previously herein and are incorporated by reference. Furthermore, Xiang teaches the selection (i.e. recovery) medium comprises 2.5 mg of 2,4-D, 600 mg casein hydrolysate, 30 g of sucrose, 100 mg of inositol, and 4.6 g of phytagel, and pH is 5.8 (Supplementary Table 2). Additionally, Yagoob teaches using 4.4 g of MS medium (p. 348, table 1), which is reasonably interpreted as 4.43 g of MS salts.
However, Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo do not explicitly teach the media comprises 6-BA and timentin, the specified concentrations of the media components, nor do they teach the pH is 5.7.
In analogous art, Wang teaches selecting transgenic rice callus on an MS-based medium comprising 0.2 g/L timentin (p. 5, p. 11, step 18). In other analogous art related to improving media for rice callus formation, Mostafiz teaches adjusting the medium to a pH of 5.7 (p. 3, section titled 2.3 Callus Induction Experiment). In other analogous art, Zaidi teaches the addition of 6-BA enhanced callus induction, embryogenic calli and regeneration (p. 568, §2).Furthermore, Zaidi teaches the importance of optimizing media by adjusting various concentrations and types of basic media, carbohydrate sources and concentrations, agar concentrations, amino acids, cytokinins and auxins is important to optimize callus induction and transformation of various rice genotypes (abstract).
It would therefore have been obvious to a person of ordinary skill in the art to modify the invention of as taught by Xiang, Jyothishwaran, Vain, Yaqoob, Ozawa, Wang, Wanichananan, and Guo to include the limitations of Wang, Mostafiz, and Zaidi to arrive at the instantly claimed method with a reasonable expectation of success because Wang, Mostafiz, and Zaidi teach media necessary for callus growth and Agrobacterium-mediated transformation. Specifically, Wang teaches selecting for transgenic callus on an MS-based media supplemented with timentin, which is an antibiotic that is routinely used and well known in the art to prevent Agrobacterium overgrowth. Mostafiz teaches successful callus growth on a media with a pH of 5.7 (p. 3) and it is also known in the art that it is important to promote callus growth following transformation. Additionally, Zaidi teaches the importance of incorporating 6-BA and also making adjustments to concentrations of the media components to optimize callus induction and transformation efficiency (abstract, p. 568, 2). One of ordinary skill in the art could incorporate the timentin, 6- BA, pH of 5.7 and make concentration adjustments without encountering any special technical obstacles. One having ordinary skill in the art would have been motivated to do so because Zaidi teaches various adjustments to concentrations can improve transformation efficiency in different rice genotypes (title, abstract), and it would therefore be prima facie obvious to include antibiotics that prevent bacterial overgrowth, include hormones that enhance callus production, optimize media by adjusting the concentrations of the components, and adjust the pH to an optimal value for nutrient uptake and callus growth to arrive at the instantly claimed method.
Response to Arguments
Applicant argues beginning on p. 4 of remarks dated 10/23/2025 the
following arguments:
Applicant respectfully disagrees with the characterization of the references and traverses the rejections. Applicant further submits that the amendments presented herein render the rejections moot. As a monocotyledonous recalcitrant species, the transformation of wild rice (Zizania latifolia) encounters three technical bottlenecks:
(1) Cell wall barrier: Thick-walled cell layers hinder the delivery of Agrobacterium T-DNA.
(2) Seed dormancy mechanism: Endosperm phenolic compounds inhibit the viability of Agrobacterium.
(3) Unstable transient expression: Exogenous genes are quickly degraded in seed tissues.
Xiang teaches the embryo tissue transformation, as shown in the title of Xiang:
Agrobacterium-Mediated High-Efficiency Genetic Transformation and Genome Editing of Chaling Common Wild Rice (Oryza rufipogon Griff.) Using Scutellum Tissue of Embryos in Mature Seeds (emphasis added).
Xiang does not teach the following patentably distinct features:
i). the wild rice is Zizania latifolia (genus: Zizania) and the Agrobacterium strain is any one selected from the group consisting of Agrobacterium strains EHA105 and LBA4404, the co- culture is conducted for 2-3 d, and the bacterial suspension has an OD value of 0.2 at a wavelength of 600 nm.
ii). 50 mg/mL kanamycin and 50 mg/mL rifampicin are added into the YEB liquid medium, and the centrifugation is conducted at 1,000 rpm to 2,000 rpm for 5 minutes (min) in step (2).
iii). The plasmid is carrying a GFP gene.
vi). The resulting single colony that was selected in part 2 was confirmed positive via PCR/gel electrophoresis.
v). the co-culture step occurred in the dark.
vi). observing an infection status of a recovered callus under a stereo fluorescence microscope, wherein the GFP gene serving as an exogenous target gene is successfully expressed in the callus of the wild rice seed if the recovered callus appears green fluorescence under ultraviolet light.
Zizania latifolia is a species of wild rice of Zizania genus, while the wild rice in Xiang is (Oryza rufipogon Griff.), and they have substantial differences, which is supported by Ning Yan et al, Chromosome-level genome assembly of Zizania latifolia provides insights into its seed shattering and phytocassane biosynthesis, COMMUNICATIONS BIOLOGY, (2022) 5:36 (Ning, submitted herewith; Abstract; page 3, right column, paragraph 1, lines 12-14).
Jyothishwaran teaches a method of introducing plasmids into Agrobacterium tumefaciens, and specifically teaches confirming the Agrobacterium transformants are positive for the plasmid vector using PCR and gel electrophoresis (see p. 771, Col. 3).
Vain teaches transformation of rice callus using a plasmid comprising a GFP gene (abstract, p. 165, Materials and Methods section titled Rice transformation), and the plasmid resulted in constitutive expression of GFP and allowed for easily detectable and screenable bright-green fluorescence in rice tissue (see abstract). Vain also teaches using an ultraviolet lamp to identify recovered GFP-expressing callus, and detection of GFP was monitored at a higher magnification using a Nikon Microphot-5A fluorescent microscope.
Yagoob teaches the co-cultivation step occurs in the dark for 2 days at 22-28°C (i.e. 25°C), then the calli are cultured on resting media in the dark at 25°C (see p. 347, section titled Agrobacterium tumefaciens strain and transformation).
Yaqoob further teaches the use of Agrobacterium strain EHA105, the culture was grown at 28 °C until the OD600 reached up to 1, and then the cells were diluted to desired final OD600 of 0.5. (see p. 347, lines 19-25; section titled Agrobacterium tumefaciens strain and transformation), and the plates containing-cultivation medium were incubated in dark for 48-72 h at 22-28 °C.
Nevertheless, Xiang, Jyothishwaran, Vain, and Yaqoob fail to teach the Agrobacterium strain LBA4404 and that the bacterial suspension has an OD value of 0.2 at a wavelength of 600 nm.
As stated above, Yaqoob teaches that the culture was grown at 28°C till OD6oo reached up to 1, and then the cells were diluted to a desired final OD6oo of 0.5 (see p. 347, section titled Agrobacterium tumefaciens strain and transformation).
However, as demonstrated in the instant application, the transient expression rates of the EHA105-positive Agrobacterium bacterial solution in callus at OD600 values of 0.02, 0.05, 0.1, and 0.5 are much lower than these rates at an OD600 value of 0.2. This decrease in the transient expression rates is shown in FIG. 3 of the instant application.
Therefore, Yagoob does not teach, disclose, or suggest the OD6oo value of 0.2 recited in instant claim 1 for further use, nor that the OD6oo of 0.2 would have improved transient expression rates relative to other OD6oo values. A person of ordinary skill in the art, on reading Yaqoob, would not have any guidance or motivation to select an OD6oo of 0.2 to use in a method of gene expression.
The above-mentioned distinguishing features are not disclosed, suggested, or taught by Xiang, Jyothishwaran, Vain, and Yaqoob. The other cited references do not cure the deficiencies of Xiang, Jyothishwaran, Vain, and Yagoob to render the claimed invention obvious.
This argument has been fully considered and is found not persuasive for
the following reason(s):
Based on the instant claims, the apparent bottlenecks appear to be overcome using already known methods of transforming other rice varieties, including methods taught by several references of the 103 rejections that are specific to transformation of recalcitrant rice varieties. Xiang teaches transforming an alternative wild rice species with EHA105, and further teaches many other instantly claimed limitations. Both EHA105 and LBA4404 are known in the art to be more virulent than other Agrobacterium strains, e.g. GV3101, therefore it would further be obvious to choose these Agrobacterium strains for transformation of a recalcitrant rice species. Furthermore, the claimed kanamycin and rifampicin concentrations are common stock solution concentrations used and generally known in the art, and are further taught by Wang referenced previously herein. Additionally, GFP is one of the most commonly used reporter genes for identifying transformants that comprise and express the GFP reporter gene, and are often observed with a microscope. It is also common and routine to PCR/gel confirm a bacterial colony for the expected plasmid before generating transformants. These limitations are overcome in the previous 103 rejections, and they are not inventive steps. It is also generally known and understood that increasing OD600 can improve transformation efficiency, therefore it would be obvious to increase the OD600 of 0.5 taught by Yaqoob in increments to optimize transformation efficiency and determine the optimal OD600 value, including to an OD600 of 0.2. In sum, the steps of the instantly claimed method are not inventive steps and combine already known methods for rice transformation. It would be prima facie obvious to combine the known methods and apply them to a known rice cultivar (Zizania latifolia) for the same purpose of Agrobacterium-mediated transformation. It is also noted that Applicant argues the wild rice species Zizania latifolia is substantially different from the wild rice species taught by Xiang. However, Applicant fails to explain why any of the listed differences in their arguments would present a special obstacle. Despite genetic dissimilarity, both species are wild rice species, and it would be obvious to apply transformation methods of one wild rice species, as well as other known and routine transformation methods of other rice cultivars including those used for other recalcitrant cultivars, and apply them to the wild rice species Zizania latifolia. For these reasons, Applicant’s argument is not found persuasive.
Applicant argues beginning on p. 6 of remarks dated 10/23/2025 the
following arguments:
Ozawa discloses establishment of a high efficiency Agrobacterium-mediated
transformation system of rice (Oryza sativa L.) (see Title of the article), and Ozawa further discloses the best conditions for co-cultivation of rice calli on solid medium are a temperature of 25°C with an Agrobacterium concentration of OD = 0.2 and 200 M acetosyringone. The Agrobacterium strain is EHA101harboring pCAMBIA1301 (see Abstract; page 523, left column, paragraph 5).
However, the present application is directed to transient transformation of a Zizania latifolia using Agrobacterium, and as stated above, the rice species in the instant application is Zizania latifolia (genus: Zizania), which is different from Oryza sativa L. (genus: Oryza) taught in Ozawa, and the transformation of wild rice (Zizania latifolia) encounters three technical bottlenecks as known in the art and listed above. One of ordinary skill in the art would not have a reasonable expectation that methods applicable to Oryza sativa L. would be successful in Zizania latifolia, and thus would not expect the teachings of Ozawa to be able to be modified for use in Zizania latifolia.
Therefore, due to the differences in rice species and Agrobacterium, combining the teachings of Xiang, Jyothishwaran, Vain, Yagoob, and Ozawa will not result in the method recited in instant claim 1.
Cho teaches stable transformation of rice (Oryza sativa L.) via microprojectile bombardment of highly regenerative, green tissues derived from mature seed.
Shiri teaches an improved Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.) cultivars, and Shiri further teaches.
Sasaki teaches the promotion of seedling growth of seeds of rice (Oryza sativa L. cv. Hitomebore) by treatment with H202 before sowing.
Zaidi teach the optimization tissue culture media for efficient transformation of different indica rice genotypes.
Mostafiz teaches efficient callus induction and regeneration in selected indica rice.
Wang teaches protocol and growth media for targeted modification of the rice genome using base editing.
Wanichananan teaches a highly efficient method for Agrobacterium-mediated transformation in elite rice varieties (Oryza sativa L. spp. indica).
Thao teaches the optimization of Agrobacterium-mediated transformation procedure for an indica rice variety.
To sum up, although Cho, Shiri, Sasaki, Zaidi, Mostafiz, Wang, Wanichananan, and Thao teach the parameters and the culture media for the treatment of the seed, they disclose the methods applicable for Oryza sativa L or indica rice, and fail to teach, disclose, or suggest the instant method for gene expression by transient transformation of Zizania latifolia using Agrobacterium (EHA105 or LBA4404). One of ordinary skill in the art would not have a reasonable expectation that methods applicable to Oryza sativa L. would be successful in Zizania latifolia, and thus would not expect the teachings of any of these references to be able to be modified for use in Zizania latifolia.
Due to the differences in rice species and treatment parameters as discussed above in more detail, there is no guidance or motivation for one of ordinary skill in the art to arrive at the method of claim 1 and have a reasonable expectation of success. Therefore, clai