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 Claims
The amendments received on 01/29/2026 have been entered. Claims 1, 3-4, 7, 13-15, 23, 25-29, and 34-37 are pending.
Claims 13-15, 23, and 25-29 remain withdrawn for being directed to a non-elected invention(s).
Claim 2 has been canceled.
Claims 1, 3-4, 7, and 34-37 are examined in this Office Action.
The text of those sections of Title 35, U.S. Code, not included in this action, can be found in a prior Office action.
Claim Rejections - 35 USC § 103
Claims 1, 3-4, 7, and 34-36 are rejected under 35 U.S.C. 103 as being unpatentable over Sa’ar (Sa’ar, 2016, The involvement of isocitrate dehydrogenase gene in tomato (Solanum lycopersicum) ripening, Master of Biotechnology Thesis, Tel-Hai College, included in IDS dated 07/05/2023) as evidenced by Gamrasni (Gamrasni, 2020, Agronomy, Vol. 10(11), pages 1-15). All dependent claims are included in these rejections unless they include a limitation that overcomes the deficiencies of the parent claim. This is a modified rejection necessitated by amendment.
Claim 1 recites “[a] plant producing a climacteric fruit, the plant comprising at least one cell comprising at least one premature stop codon mutation in a cytosolic isocitrate dehydrogenase 1 (ICDH1) encoding gene, wherein the plant has a reduced ethylene production rate compared to control, a reduced respiration rate compared to control, or both, wherein said premature stop codon mutation results from a frameshift mutation that produces a non-functional truncated ICDH1 protein less than half the size of wild-type ICDH1”.
Sa’ar teaches silencing the ICDH gene (i.e., premature stop codon in a cytosolic isocitrate dehydrogenase 1 (ICDH1) encoding gene) in Microtom and DEL ROS1 tomatoes (i.e., a plant producing a climacteric fruit) via virus induced gene silencing (VIGS).
Sa’ar teaches the ethylene control motif (CCGCGT) found in the ICDH gene located in the 5’ UTR region (Sa’ar, page 9, Section 1.7, last paragraph and page 10, Figure 9). The SlICDH1 gene (i.e., a cytosolic isocitrate dehydrogenase 1 (ICDH1) encoding gene) contains the AtSR1 binding motif (A/C/G)CGCG(G/T/C)1.
Sa’ar teaches that Microtom tomatoes in which the ICDH gene was silenced produced less ethylene during the test period compared to the control tomatoes (i.e., wherein the plant has a reduced ethylene production rate compared to a control) in which the gene was not silenced (empty) (Sa’ar, page 38, Section 3.4.1.1).
Sa’ar teaches that the goal of his research was to find the connection between the effect of ethylene and climactic respiration through knocking out the ICDH gene (Abstract, page 65, third paragraph). Sa’ar teaches knocking out the ICDH gene using virus induced gene silencing (VIGS). Microtom and DEL ROS1 tomatoes were infected with a plasmid carrying ICDH in order to silence its expression (ICDH-) (Sa’ar, page 38, Section 3.4). Additionally, Sa’ar suggests creating a stable transgenic plant with different silencing levels and/or using CRISPR technology to create a gene mutation in the ICDH gene, and examining the effects of the mutation on fruit ripening (Sa’ar, page 49, Summary 4.5).
Although Sa’ar does not explicitly teach introducing a premature stop codon mutation in the ICDH gene, it is well known in the art that a premature stop codon mutation is just one of many types of mutations capable of gene silencing that one having ordinary skill in the art would consider as an obvious substitution to achieve similar results. Thus, introducing a premature stop codon mutation is obvious and is merely a design choice.
At the time the instant application was filed, it would have been obvious and within the scope of one of ordinary skill in the art to reduce the expression of the ICDH1 gene in order to reduce the ethylene production and the respiration rate to delay the ripening and increase the shelf life of a climacteric fruit, as taught by Sa’ar. One would have been motivated to introduce a mutation in the ICDH1 gene knowing that it would reduce the ethylene production and the respiration rate thereby delaying ripening and increasing the shelf life of a climacteric fruit, as taught by Sa’ar. Thus, one of ordinary skill in the art would have a high expectation of success by following the teachings of Sa’ar.
The method of introducing a mutation in a gene (including a premature stop codon) to reduce its expression is a technique that was routine in the art at the time the application was filed, as taught by the cited references and the state of the art in general.
In regard to claim 3, Sa’ar teaches Microtom and DEL ROS1 control tomatoes were infected with a plasmid without an insert (empty) in which the ICDH gene was not silenced (i.e., said control is a genetic reference plant comprising the same genotype of said plant and is lacking said mutation) (Sa’ar, page 38, Section 3.4.1.1).
In regard to claim 4, Sa’ar teaches in DEL ROS1 tomatoes, the expression levels of the ICDH gene following the silencing decreased significantly compared to the control (empty) in all ripening conditions (i.e., the cytosolic ICDH1 comprising said mutation has a reduced NADP-dependent ICDH activity compared to a control ICDH) (Sa’ar, page 40, Section 3.4.2.1).
In regard to claims 7 and 34-35, Sa’ar teaches the effect of silencing the ICDH gene in Microtom tomatoes (i.e., a plant part selected from the group consisting of: a fruit, a flower, a seed, and a leaf; wherein said plant part is a fruit) resulted in them reddening more slowly compared to the control tomatoes. In the control tomatoes, a red color began to appear after about 3 days from the beginning of the tests. In ICDH tomatoes, the appearance of the red color was delayed for 11 days, and appeared only after about 14 days (i.e., wherein said fruit has a developing to ripening period at least 10% longer than a fruit derived from a genetic reference plant) from the start of the test (Sa’ar, page 39, Section 3.4.1.2).
In regard to claim 36, Sa’ar teaches that by inhibiting the expression of the ICDH gene, a delay in ethylene production and the ripening of fruit was found. The delay was expressed in ripening indicators (ethylene production and color change), and accordingly extended the shelf life of the fruit (i.e., having a shelf life increased by at least 7 days) (Sa’ar, page 49, first paragraph).
In regard to the ripening indicator of ethylene production, ethylene production in the control tomatoes was higher than in the ICDH- tomatoes for 12 days (i.e., having a shelf life increased by at least 7 days compared to a plant part derived from a genetic reference plant). Ethylene production peaked on the eighth day in the control compared to the fifteenth day for the ICDH- tomatoes, and the peak height was more than double in the control compared to the ICDH- (see Fig. 33 below) (Sa’ar. Page 42, Section 3.4.2.2).
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Fig. 33 (page 42) Ethylene production in the DEL ROS1 tomato; empty (tomatoes that were infected and the gene was not silenced); ICDH (tomatoes that were infected and the gene was silenced).
In regard to the ripening indicator of color change, the change to a red shade was delayed in the ICDH DEL ROS1 tomatoes relative to the control (see Figs. 34 and 35 below) (Sa’ar, page 42, Section 3.4.2.3).
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Figs. 34 and 35 (page 43) Tomato peel color DEL ROS1; empty (tomatoes that were infected and the gene was not silenced); ICDH (tomatoes that were infected and the gene was silenced).
Claim 37 is rejected under 35 U.S.C. 103 as being unpatentable over Sa’ar in view of Sulpice (Sulpice et al., 2010, Amino Acids, Vol. 39, pp. 1055–1066; included on IDS dated 07/05/2023). This is a new rejection necessitated by amendment.
Claim 37 recites “[t]he plant of claim 1, wherein said control comprises a plant wherein said cytosolic ICDH1 gene is silenced by an RNA interfering (RNAi) encoding polynucleotide”.
Sa’ar teaches the plant of claim 1.
Sa’ar does not explicitly teach wherein said control comprises a plant wherein said cytosolic ICDH1 gene is silenced by an RNA interfering (RNAi) encoding polynucleotide.
Sulpice teaches that transgenic tomato (Solanum lycopersicum) plants were generated targeting the cytosolic NADP-dependent isocitrate dehydrogenase gene (SlICDH1) (i.e., ICDH1 gene) via the RNA interference approach (i.e., an RNAi encoding polynucleotide) (Sulpice, Abstract).
Twenty-five tomato plants underwent Agrobacterium tumefaciens-mediated transformation. A first screening of the lines was performed by measuring total cellular ICDH activity. These preliminary studies allowed the identification of two lines that showed a statistically significant and specific reduction in ICDH1 expression and total ICDH activity in leaves. These two transgenic lines were clonally propagated. Following a period of 5 weeks growth, leaves were harvested, and total ICDH and NAD-dependent IDH activities were measured in order to confirm the reduced activity of NADP-dependent ICDH and its effects on total NAD-dependent IDH activity. To verify the specificity of the constructs, a secondary screen was performed at the mRNA level. This revealed that only SlICDH1 expression was significantly reduced in the transgenic lines. When taken together, these results demonstrate that the two lines are suitable for assessing the transcriptional, biochemical, and physiological effects of a mild reduction of the cytosolic ICDH isoform activity (control comprises a plant wherein said cytosolic ICDH1 gene is silenced by an RNAi encoding polynucleotide) (Sulpice, Results, page 1056, right column, second paragraph).
Although Sulpice does not explicitly teach a plant wherein said cytosolic ICDH1 gene is silenced by an RNA interfering (RNAi) encoding polynucleotide used as a control, it would have been obvious and within the scope of one of ordinary skill in the art to use a plant wherein said cytosolic ICDH1 gene is silenced by an RNA interfering (RNAi) encoding polynucleotide as a control, by combining the teachings of Sa’ar and Sulpice. One would have been motivated to use a plant wherein said cytosolic ICDH1 gene is silenced by an RNA interfering (RNAi) encoding polynucleotide as a control knowing that it is suitable for assessing the transcriptional, biochemical, and physiological effects of a mild reduction of the cytosolic ICDH isoform activity, as taught by Sulpice. Thus, one of ordinary skill in the art would have a high expectation of success by following the teachings of Sa’ar and Sulpice.
Response to Applicant’s Arguments
Applicant's arguments filed 01/29/2026 have been fully considered but they are not persuasive.
Applicant argues that the premature stop codon mutation produces unexpectedly superior results compared to VIGS silencing, supporting the non-obviousness of the claimed invention.
The Examiner respectfully disagrees. Sa’ar states that to understand the involvement and importance of the ICDH gene in tomato ripening, the gene was silenced using the VIGS method (page 38). In regard to virus-induced gene silencing (VIGS), YING (Ying et al., 2024, Journal of Integrative Agriculture, Vol. 23(5), pp. 1557-1567) teaches that it is an efficient method for verifying gene function through the induced rapid knockdown of target genes and the generation of physiological and phenotypic variations at the transcription level in host plants. The benefits of this technology include easy manipulation, a short culture cycle, and high silencing efficiency (Ying, Introduction, first paragraph). YING further teaches that CRISPR/Cas systems are newly developed third-generation artificial endonuclease systems used to generate DNA double-strand breaks (DSBs) in the genome. In crop plants, gene editing techniques enable precise genomic modifications, including insertion, deletion, and substitution. CRISPR/Cas9 technology has been successfully applied for improving resistances and abiotic stress tolerances in several plants (Ying, Introduction, second paragraph). Given that VIGS is a rapid, transient, and non-heritable technique that is generally used to study the effects of gene silencing, it is not at all unexpected that a permanent, precise gene-editing tool that knocks out or modifies the DNA sequence (such as CRISPR) would lead to superior results.
Furthermore, Sa’ar states “In conclusion, inhibition of the expression of the ICDH gene, which encodes a key enzyme in the TCA cycle, was found to delay the production of ethylene and fruit ripening. The delay was expressed in ripening indices (ethylene production and color change) and accordingly extended the shelf life of fruit. This is a new way to delay and control the ripening process using a gene from cellular respiration”.
Applicant’s affidavit cited the following figures for comparison. Figure 1a is taken from Sa’ar, showing the Micro-Tom tomato peel color change (Sa’ar, Figure 31, page 40); Figure 1b shows the peel color change in an undisclosed variety of tomato (Affidavit, page 4); and Figure 35 was taken from Sa’ar, showing the peel color change in the DEL ROS1 variety of tomato (Sa’ar, Figure 35, page 43), which aligns almost perfectly with Figure 1b.
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Figure 35. DEL ROS1 tomato skin color according to parameter *a. The tomatoes
were kept at 20°C. Solid line – control, empty, dotted line – ICDH.
Additionally, Applicant argues that color development is a direct downstream indicator of
ripening that is affected by ethylene production and respiration. Figure 6A is taken from the instant Specification; Figures 29 and 33 are taken from Sa’ar. Again, the ethylene production graphing of the DEL ROS1 tomato aligns almost perfectly with that of instant Figure 6A.
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(Sa’ar, Figure 29, page 39, Micro-Tom)
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(Sa’ar, Figure 33, page 42, DEL ROS1)
Thus, Sa’ar teaches that inhibition of the expression of the ICDH gene was found to delay the production of ethylene and fruit ripening. Sa’ar teaches the results of silencing the ICDH1 gene on ethylene production and peel color change in the Micro-Tom and DEL ROS1 tomatoes. In both respects, the ethylene production and peel color change in the DEL ROS1 tomato aligned very closely with the results in the Affidavit as well as the instant Specification.
Summary
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Correspondence
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CHRISTINA MEADOWS whose telephone number is (703)756-1430. The examiner can normally be reached Monday - Friday 9:00 am - 5:00 pm.
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CHRISTINA MEADOWS
Examiner
Art Unit 1663
/CHRISTINA L MEADOWS/Examiner, Art Unit 1663
/Amjad Abraham/ SPE, Art Unit 1663
1 As evidenced by Gamrasni (Gamrasni, 2020, Agronomy, Vol. 10(11), pages 1-15; see page 3, Materials and Methods, Section 2.6).
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