In planta genetic transformation to produce CRISPRed high-oleic peanut

In planta genetic transformation to produce CRISPRed high-oleic peanut | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In planta genetic transformation to produce CRISPRed high-oleic peanut Hong Wei Han, Shu Tao Yu, Zhi Wei Wang, Zhen Yang, Chun Jiao Jiang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1096211/v2 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Jun, 2023 Read the published version in Plant Growth Regulation → Version 2 posted 5 You are reading this latest preprint version Show more versions Abstract In contrast to its normal-oleic counterpart, high-oleic peanut has better keeping quality and multiple health benefits. Breeding high-oleic peanut through conventional means is a tedious process generally requiring several years. Genome editing, however, may shorten the duration. In this study, node injection method was used to transform normal-oleic Huayu 23, a popular peanut cultivar having dysfunctional FAD2A and functional FAD2B , with CRISPR/Cas9 construct targeting FAD2B , and two T0 seeds with over 80% oleic acid and 442A insertion in FAD2B were obtained. A T1 plant grown from the viable T0 seed produced high-oleic seeds. As a genotype-independent, simple and easy method for peanut genetic transformation, node injection has great potential in functional analysis of genes and peanut varietal improvement. Groundnut Arachis Genome editing High oleate bar Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Rich in culinary oil and highly digestible protein, the cultivated peanut ( Arachis hypogaea L.) occupies an important position in human and animal nutrition(Li et al. 2022 ). Fatty acid profile is an indicator of its quality. Oleic and linoleic acids together constitute about 80% of total fatty acids in peanut seeds. As compared to linoleic acid, oleic acid is less prone to oxidation. Increase in oleic acid and decrease in linoleic acid in peanut seeds may result in extended shelf life of peanut produce and much more health benefits (Nkuna et al. 2021 , Zhao et al. 2022 ). In the cultivated peanut, FAD2A and FAD2B control the conversion of oleic acid to linoleic acid, and expression of the high-oleic phenotype (at least 70% oleic acid content) in peanut cultigen requires inactivation of both genes (Nawade et al. 2018 ). Natural, chemical, and physical peanut mutants with high oleate have been reported and used in hybridization and backcross to develop high-oleic peanut cultivars (Wang et al. 2021 )(Wang et al. 2022 )(Han et al. 2022 ). In contrast to the lengthy process of conventional breeding, the speed of genome editing may be much faster. Using peanut germs with a cotyledon attached for transformation, Wen et al. ( 2018 ) demonstrated that TALENs-mediated targeted mutagenesis of FAD2 in peanut cv Yueyou 7 raised oleic acid content from 43–60%~80%, while decreased linoleic acid content from 35.5% to lower than 20% (Wen et al. 2018 ). Yuan et al. ( 2019 ) induced FAD2B mutations in peanut protoplasts and hairy roots using CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 based genome editing. Zhang et al. ( 2021 ) bombarded the embryonic calli of peanut cv Luhua 11 with a CRISPR/Cas9 gene editing vector targeting FAD2 , and regenerated plants were obtained, but the fatty acid profiles of the descendants were not reported (Zhang et al. 2021 ). Tang et al. ( 2022 ) produced gene edit Huayu 23 lines with decreased saturated fatty acid and increased unsaturated fatty acid through knockout of FATB ( Acyl-acyl carrier protein thioesterase B ) by CRISPR/Cas9 system. Suitable transformation procedures may facilitate the application of genome editing tools. Previously, node injection method, an easy-to-follow in planta peanut transformation protocol, was developed at our laboratory (Wang et al. 2013 ). It was used to transfer soybean SCTF-1 gene to chill susceptible peanut, RT-PCR and Southern blot analysis confirmed the transgenic events. Peanut plants with SCTF-1 gene showed good tolerance to chill stress (V. M. Vacu, unpublished data). Likewise, transfer of AhCYP , a bacterial wilt infection responsive gene (Ding et al. 2012 ) to Huayu 40, a susceptible peanut cultivar, enhanced bacterial wilt resistance of the recipient (吴琪 et al. 2019). This study aimed to test its effectiveness in inducing high-oleic peanut mutations using CRISPR/Cas9 technology. Material And Methods Plant material for genome editing and its cultivation Huayu 23, a popular peanut normal-oleic cultivar of runner market type widely accepted by growers and food processors in China, was used in this study. As expected, Sanger sequencing of its FAD2A and FAD2B and subsequent sequence alignment revealed that this cultivar had a mutated FAD2A (448 G > A) and a wild type FAD2B . Peanut for genome editing was sown under polyethylene film mulching in an isolated region in SPRI Laixi Experimental Station on May 5, 2021. Agronomic practices were followed as routine. Vector construction and transformation of Escherichia coli Target site of sgRNA, 501–520 position of the coding sequence (5’-catgaacaatccaccaggga-3’) of wild type FAD2B , was selected using CRISPR-GE ( http://skl.scau.edu.cn/ ) (Fig. 1 ). To facilitate ligation in genome editing vector construction, two oligos which would produce overhangs after mixing, denaturation and annealing were generated with online tool ( http://biogle.cn/index/excrispr ) and synthesized (Tsingke, Qingdao) (FAD2B-1F: 5’-gggttgcatgaacaatccaccaggga-3’, FAD2B-1R: 5’-aaactccctggtggattgttcatgca-3’). CRISPR/Cas9 editing vector with target site incorporated was made with BGK41-Cas9 (Biogle Biotechnology Co. Ltd, Hangzhou) following manufacturer’s instructions. Briefly, BGK41-Cas9, oligo dimers and enzyme mix (Biogle CRISPR/Cas vector construction kit) were mixed on ice bath, and then incubated at 20 ℃ for 1 h. The ligation products were transformed into competent cells of E. coli strain DH5α. Positive clones identified using colony-PCR with Cas9-F/Cas9-R primer pair (5’-tcgtgctgaccctgacactgtttga-3’, 5’- cttggcggtagccttgccgatttcc-3’) were sequenced with primer CXYW1 (5’- cccagtcacgacgttgtaaa-3’) (Tsingke, Qingdao) to confirm the inclusion of the target site in the vector. The newly constructed plasmid was named as BGK41-Cas9 recombinant vector FAD2B-1 (Fig. 1 ) Transformation of Agrobacterium and node injection transformation of peanut Genome editing construct was transformed into Agrobacterium tumefaciens strain GV3101 chemically competent cells (Veidi Biotech, Shanghai) according to the attached user’s guide. Preparation of Agrobacterium for injection was based on Pan et al. ( 2020 ) with some modifications. Positive single clones were verified by bacterial suspension PCR using Cas9-F/Cas9-R primer pair. 100 µl of freshly prepared bacterial suspension were cultured in 10 ml of YEB liquid medium at 28 ℃ with agitation (250 rpm) until OD 600 reached 0.6–0.8 (about 12 h). The cultures were centrifuged at 6 000 rpm for 1 min to collect the bacterial cells. Equal volume of infection solution containing 100 µmol/L acetosyringone (BBI Lifesciences, Hongkong), 10 mmol/L MES (Sangon Biotech, Shanghai) and 10 mmol/L MgCl 2 ·6H 2 O was added to the pellets. Resuspended bacterial pellets were used for injection. Node injection procedure was essentially the same as that in our previous report except for the plant age (Wang et al. 2013 ) (Fig. 2 ). Injection was done between 6:00–8:00 a.m. on July 17, 2021, and the positions injected were marked with threads (Fig. 2 ). Inverted U-shaped mental wires were used to facilitate the entry of pegs into the soil from higher nodes (Fig. 2 ). Fatty Acid Analysis Of Resultant T0 Seeds Pods were harvested when matured (Sept. 17, 2021). These pods were sun-dried and hand shelled. Oleic and linoleic acid contents of the individual single seeds were predicted with NIRS (Wang et al. 2014 ). Seeds with at least 74% oleic acid along with the untreated control Huayu 23 were further analyzed for fatty acids by gas-chromatography using cotyledonary slices follow the protocol of Yang et al. ( 2012 ). Comparison of FAD2 sequences between the high-oleic T0 seeds and Huayu 23 FAD2A and FAD2B sequences of the high-oleic T0 seeds and Huayu 23 were amplified by PCR using primers aF19 (5’-gattactgattattgactt-3’)/R1 (5’- ctctgactatgcatcag-3’) and bF19 (5’-cagaaccattagctttg-3’)/R1, respectively (Patel et al. 2004 ), and DNA templates prepared from cotyledonary slices (Yu et al. 2010 ). PCR products were directly sequenced by Tsingke, Qingdao. Sequence comparison was done with the DNAStar Lasergene version 7.1.0. Amplification of bar gene PCR amplification of bar gene were performed using DNA templates prepared from Cas9 empty vector (positive control), Huayu 23 (negative control) and 2 high-oleic T0 seeds respectively, with Bar-7F and Bar-6 primer (Bar-7F: 5’-caccatcgtcaaccactaca-3’,bar-6r༚5’-acttcagcaggtgggtgta-3’). The PCR mixture (10 µ1) consisted of 6.25 µ1 of 2× Taq Plus Master Mix Ⅱ (Vazyme, Najing), 1 µ1 of DNA template, 0.5 µ1of primers (10 µM) each, and 4.25 µ1 of double distilled water. PCR thermal profile was 95 ℃ 5 min, followed by 30 cycles of 95 ℃ for 40 s, 61 ℃ for 40 s, and 72 ℃ for 30 s, and a final extension of 72 ℃ for 10 min. Cultivation Of T1 Plants To obtain descendants as soon as possible, one T0 seed was sown in a pot in the winter of 2021, but unfortunately it died and did not set any pods. The remaining T0 seed along with a Huayu 23 seed was sown under film mulch on May 23, 2022. Both grew to maturity and were harvested on September 20, 2022. Fatty Acid Profiling Of The T2 Seeds Harvested From T1 Plant Oleic and linoleic acid contents of the T2 seeds from the T1 plant both as bulk seed sample and as individual single seeds were determined by NIRS (Wang et al. 2021 , Wang et al. 2014 ). Huayu 23 CK was also analyzed for main fatty acid content. Results And Analysis Quality of the resultant T0 seeds and wild type Huayu 23 predicted by NIRS A total of 16 nodes were injected, which only resulted in 4 seeds. Among them, two seeds, Huayu 23-7-1 and Huayu 23-7-2, were classified as high-oleic by NIRS (Table 1 , Fig. 3 ). Table 1 Chemical quality of single T0 peanut seeds and Huayu 23 predicted by near infra-red spectroscopy Identity Generation Oleic acid (O) (%) Linoleic acid (L) (%) O/L Huayu 23 (CK) - 45.88 26.64 1.72 Huayu 23-7-1 T0 74.33 5.39 13.80 Huayu 23-7-2 T0 78.96 3.84 20.54 Main fatty acids of the resultant seeds and wild type Huayu 23 by wet chemistry method Fatty acid composition of the seeds of concern determined by GC was shown in Table 2 . There were drastic changes in oleic, linoleic and palmitic acid contents in Huayu 23-7-1 and Huayu 23-7-2. Oleic acid increased from 52.15% in Huayu 23 to over 80% in Huayu 23-7-1 and Huayu 23-7-2, linoleic acid dropped from 25.72% in Huayu 23 to lower than 1.70% in Huayu 23-7-1 and Huayu 23-7-2. Accordingly, the oleic acid to linoleic acid ratio (O/L) rose from around 2.03 to more than 47. Palmitic acid content in Huayu 23-7-1 and Huayu 23-7-2 was 8.07% and 7.39%, respectively, much lower than that in Huayu 23 (15.55%). Table 2 Fatty acids (%) in single peanut seeds determined by gas chromatography Identity Generation Oleic acid Linoleic acid Palmitic acid O/L Huayu 23 (CK) - 52.15 ± 0.24B 25.72 ± 0.20A 15.55 ± 0.19A 2.03 Huayu 23-7-1 T0 80.26 ± 0.32A 1.68 ± 0.12B 8.07 ± 0.23B 47.69 Huayu 23-7-2 T0 80.83 ± 0.10A 1.46 ± 0.06B 7.39 ± 0.38B 55.36 In each column of the fatty acid content, figures followed by the same letter were not significantly different at 0.01 level. Fatty acid content was expressed as mean ± SE. FAD2A/FAD2B Genotyping of the resultant T0 seeds and wild type Huayu 23 Multiple alignment of FAD2A/FAD2B sequences of different sources revealed that Huayu 23, Huayu 23-7-1 and Huayu 23-7-2, all had a mutated FAD2A (448 G > A), Huayu 23 possessed wild type FAD2B , whereas both the high-oleic T0 mutants seeds had a mutant type FAD2B (442A insertion) (Fig. 4 ). The causal relationship between 442A insertion in FAD2B and dysfunctional FAD2B has been well clarified (Yu et al. 2008 ). However, the 442A FAD2B mutation was out of the scope of the anticipated targeting site, 501–520 position of the coding sequence of wild type FAD2B , where no mutation was detected. Amplification of bar gene As expected, the 2 high-oleic peanut seeds produced a band with size equal to the positive control Cas9 empty vector, whereas the untreated Huayu 23 (negative control) yielded no band (Fig. 5 ), verified that the 2 high-oleic peanut seeds were transformants. T1 Plants Grown From High-oleic T0 Seeds The two high-oleic T0 seeds along with the untransformed control were sown. Both T0 seeds developed into plants, but one died prior to flowering (Huayu 23-7-2). The remaining T1 plant (Huayu 23-7-1) grew normally and set a total of 73 seeds. Oleic And Linoleic Acid Contents Of T1 Plant All the seeds from the T1 plant Huayu 23-7-1 were firstly used as bulk seed sample in NIRS. The T1 plant had an oleic acid content of 79.07%, as against 44.52% in Huayu 23. Then sixty-eight well-developed T2 seeds were analyzed with NIRS for individual single seeds, and all were found to be high-oleic (Table 3 ). Likewise, forty-five Huayu 23 seeds were also used as individual single seed samples in NIRS analysis and, as expected, all were normal-oleic (Table 3 ). Compared to Huayu 23, the T1 plant was shorter and set slightly bigger pods (Fig. 6 , Table 4 ). Pod and seed weight of the T1 plant was also much higher (Table 4 ). Since the T1 plant and its parent were cultivated apart to avoid possible mechanical and biological mixing, it is still unknown these were caused by environmental factors or not. Table 3 Chemical quality of bulk and single T2 peanut seeds and Huayu 23 predicted by NIRS Identity Generation Bulk/Single Oleic acid (%) Linoleic acid (%) O/L Huayu 23 (CK) - Bulk 44.52 35.99 1.24 Huayu 23-7-1 T2 Bulk 79.07 5.01 15.80 Huayu 23 (CK) - Single 37.82 ± 0.97 37.25 ± 0.61 1.04 ± 0.04 Huayu 23-7-1 T2 Single 75.60 ± 0.33 5.67 ± 0.26 22.91 ± 7.20 Fatty acid content and O/L were expressed as mean ± SE, where appropriate. Table 4 Main agronomic characters of gene-edited Huayu 23 T1 plant and Huayu 23 Plant identity Main stem height (cm) Length of cotyledonary branches (cm) Stem thickness (cm) Rang of pod-bearing branches (cm) Number of branches Number of effective branches Number of pods Pod weight (g) Seed weight (g) Huayu 23 (CK) 37 40 0.5 10 9 9 53 44.18 27.92 Huayu 23-7-1 25 33 0.5 8 19 17 59 61.00 36.75 Discussion It seems that biallelic genome editing in this report is questionable. In fact, in a separate study, using a novel technology and with the help of NIRS for bulk seed samples, we were able to obtain high-oleic peanut M1 chemical mutant single plants from a popular normal-oleic Spanish market type cultivar with wild type FAD2A and wild type FAD2B . In other words, it appeared that in these high-oleic mutants, not only were both FAD2A and FAD2B mutated, but that these mutations were homozygous (C. T. Wang, unpublished data). This may be ascribed to targeting reproductive cells at earlier stages rather than postzygotic tissues at later stages. The possibility that the mutant FAD2B allele was in the background genotype of the cultivar used for transformation can be fully excluded. To be on the safe side, special care was taken to use breeder seeds, and the olei acid phenotype and FAD2A/FAD2B genotype of Huayu 23 used for transformation were ascertained prior to the experiment. In this study, evidence from fatty acid profile of the T0 and T2 seeds, FAD2B genotyping and bar gene amplification all supported that genome editing of Huayu 23 was successful. Two peanut mutant T0 seeds with over 80% oleic acid were generated via CRISPR/Cas9 genome editing technology following the node injection method developed by Wang et al. ( 2013 ) (Wang et al. 2013 ), and the high-oleic phenotype was expressed in T2 seeds clearly verified the usefulness of the peanut transformation protocol. The rationale behind the node injection method is that most of the peanut seeds set on the first (cotyledonary branches) and second pairs of branches, the possibility of harvesting sound mature kernels from the lower nodes was high, and that peanut cells that will develop into reproductive cells, or “primordial” reproductive cells, can be transformed (Wang et al. 2013 ). Generally, only the first and second nodes counting from the intersection of the main stem and cotyledonary branches were injected at 30 days after sowing (Wang et al. 2013 ). However, in this study, when everything was ready, it was too late (63 days after sowing), only higher nodes could be injected. That is the reason why only a small number of seeds were harvested. Failing to edit in the anticipated target site may be due to the small population and/or the unsuitable oligos designed for genome editing vector construction, as the website for oligo design had no peanut genome option. Anyway, two CRISPRed high-oleic peanut seeds were identified from the 4 resultant seeds. Changes in oleic and linoleic acid contents in T0 and T2 seeds and the FAD2B sequences (442A) of the two peanut T0 transformants demonstrated that the high-oleic phenotype was inheritable. In addition to high-oleic acid phenotype, high and stable productivity is a prerequisite for a peanut cultivar to be accepted by growers. It is still necessary to evaluate the overall performance of the derived lines in due course if commercialization is considered. In peanut, pods develop from pegs. One or multiple peg(s) is/are born at each node with peg(s), depending on cultivar (Nigam et al. 1990 ). In the case of multiple pegs, one injection may result in more than one pods, and in the meantime, the “primordial” reproductive cells at different developmental stages may increase the chances of being transformed. In this regard, node injection method is advantageous over flower injection. Our earlier study indicated that the method was genotype independent (Wang et al. 2013 ), where no tissue culture procedure was needed, expanding its scope of use. Nevertheless, we believe that in-depth developmental studies may help optimize peanut transformation efficiency of this method. Since the peanut node injection transformation method is easy to implement, and as peanut is an oilseed crop and its seeds can be eaten raw, the node injection method may facilitate peanut molecular pharming. It is anticipated that the method, coupled with genome editing technology where necessary, will find wide utility in areas such as functional analysis of candidate peanut genes and development of genome edited peanut cultivars with improved safety quality, ideotype, and high and stable productivity. This node injection transformation method is not only useful to peanut, but also of some reference to other seed plant species. Conclusions To sum up, using the node injection transformation method and a CRISPR/Cas9 construct targeting FAD2B , two high-oleic peanut mutant seeds with 442A insertion in FAD2B were generated from normal-oleic cultivar Huayu 23 already having dysfunctional FAD2A . Amplification of bar gene verified that the 2 high-oleic peanut seeds were true transformants. One of the T0 seed developed into a healthy plant. NIRS analysis demonstrated that the high-oleic phenotype was expressed in T2 seed generation. Abbreviations FAD2A fatty acid desaturase 2A FAD2B fatty acid desaturase 2B GC gas-chromatography MES 2-(N-morpholino) ethanesulfonic acid sodium salt NIRS near infra-red spectroscopy OD optical density TALENs transcription activator-like effector nucleases YEB yeast extract broth Declarations Ethical Statement This article does not contain any studies involving human participants or animals performed by any of the authors. This study was approved by the ethical committee of Shandong Peanut Research Institute. Informed consent was obtained from all the authors of this article. Conflict of interest All of the authors of “ In planta genetic transformation to produce CRISPRed high-oleic peanut” here declare that there is no conflict of interest. Author contributions CTW designed and supervised the experiment. HWH and ZWW conducted the transformation experiment. ZY, CJJ, XZW and XSS were responsible for peanut cultivation, NIR analysis, and regent supplies. STY performed GC analysis. CTW, HHW, and STY prepared the manuscript. Acknowledgments We express our sincere thanks to the financial support from Taishan Industry Leading Talents Special Fund (LJNY201808), Guangdong Program for Science & Technology Plan (2020B020219003), China Agricultural Research System (CARS-13), and Agricultural Science & Technology Innovation Project of Shandong Academy of Agricultural Sciences (CXGC2022A03). References Ding YF, Wang CT, Tang YY et al (2012) Isolation and analysis of differentially expressed genes from peanut in response to challenge with Ralstonia solanacearum. 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ZL 2019 1 0820261.0 Cite Share Download PDF Status: Published Journal Publication published 25 Jun, 2023 Read the published version in Plant Growth Regulation → Version 2 posted Reviewers agreed at journal 03 Dec, 2022 Reviewers invited by journal 30 Oct, 2022 Editor invited by journal 20 Oct, 2022 Editor assigned by journal 20 Oct, 2022 First submitted to journal 18 Oct, 2022 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1096211","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[{"code":1,"date":"2021-11-30 20:18:53","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research 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Wang","email":"","orcid":"","institution":"SPRI: Shandong Peanut Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhi","middleName":"Wei","lastName":"Wang","suffix":""},{"id":158809225,"identity":"11a39500-8feb-44f2-a511-4f6dbcbca6a4","order_by":3,"name":"Zhen Yang","email":"","orcid":"","institution":"SPRI: Shandong Peanut Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhen","middleName":"","lastName":"Yang","suffix":""},{"id":158809226,"identity":"0879f008-bb85-46c2-bf05-6f112e5f9524","order_by":4,"name":"Chun Jiao Jiang","email":"","orcid":"","institution":"SPRI: Shandong Peanut Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chun","middleName":"Jiao","lastName":"Jiang","suffix":""},{"id":158809227,"identity":"5b2f6bde-14ef-426a-b446-f2b148e34346","order_by":5,"name":"Xiu Zhen Wang","email":"","orcid":"","institution":"SPRI: Shandong Peanut Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiu","middleName":"Zhen","lastName":"Wang","suffix":""},{"id":158809228,"identity":"4b7e4734-95f5-43d9-937b-a5fe5ddfe2e8","order_by":6,"name":"Xiu Shan Sun","email":"","orcid":"","institution":"SPRI: Shandong Peanut Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiu","middleName":"Shan","lastName":"Sun","suffix":""},{"id":158809229,"identity":"51d7f459-e85e-437c-ab91-ef48c6fd9e0b","order_by":7,"name":"Chuan Tang Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYDACCcYGGJPxwQMD0rQwMxskEKcFzmJmk0ggRgf/7Oa2Bz932MibS+Qfq0goOCzHwH72AH5L7hxsN+w9k2a4c0Yy240Eg8PGDDx5+C0zkEhsk+BtO8y44QZYS1pigwQPfh+BtEj+bftvD9JSANRST5QWad62A4kgLQwJBjYJDIS0SNwAapE9k5y84cxjYwmgFsM2nhz8WvhnpD+TfLvDznbD8cSHHz78kZDnZz9DROwgEgAQsBFWj65lFIyCUTAKRgE6AAA8bELvPESMEwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8748-675X","institution":"SPRI: Shandong Peanut Research Institute","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Chuan","middleName":"Tang","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2021-11-19 14:08:18","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-1096211/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-1096211/v2","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10725-023-01031-y","type":"published","date":"2023-06-26T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":30219281,"identity":"3bea3095-a4c6-4351-af9c-fcbc174ccc83","added_by":"auto","created_at":"2022-12-12 17:55:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":363733,"visible":true,"origin":"","legend":"\u003cp\u003eMap of BGK41-Cas9 recombinant vector FAD2B-1 (Adapted from http://biogle.cn/index/excrispr)\u003c/p\u003e\n\u003cp\u003eLB：T-DNA right border；RB：T-DNA left border； U6：Soybean U6 promoter；SG：sgRNA scaffold；e35S：enhanced 35S 35S promoter；Cas9：Optimized Cas9；NOS Ter：NOS terminator；35S：CaMV (cauliflower mosaic virus) 35S promoter；Bar：Barsta selection marker gene；PolyA Ter：PolyA terminator\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-1096211/v2/1c7fb6533d8e29cb24b331ec.png"},{"id":30219282,"identity":"e3557662-29fa-42ed-88e8-aba9ba1aa8b0","added_by":"auto","created_at":"2022-12-12 17:55:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2257821,"visible":true,"origin":"","legend":"\u003cp\u003eNode injection of \u003cem\u003eAgrobacterium\u003c/em\u003e suspension (a) and use of inverted U-shaped mental wires to help soil penetration of pegs (b)\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-1096211/v2/ed8d57c104b6e8e9c747d1d6.png"},{"id":30219286,"identity":"4668856c-a1d1-4a15-be8f-0a87f4f07128","added_by":"auto","created_at":"2022-12-12 17:55:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":253759,"visible":true,"origin":"","legend":"\u003cp\u003eTwo high oleic peanut seeds, Huayu 23-7-1(left), Huayu 23-7-2 (middle) and wild type Huayu 23 (right)\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-1096211/v2/6e10b714ff811cde98c019bf.png"},{"id":30219283,"identity":"fc38dc51-0d9c-4d82-81dc-3253fb1fb457","added_by":"auto","created_at":"2022-12-12 17:55:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1076752,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple\u003cstrong\u003e \u003c/strong\u003esequence alignment of \u003cem\u003eFAD2A\u003c/em\u003e and \u003cem\u003eFAD2B\u003c/em\u003e from FAD2A V1.0\u003cem\u003e \u003c/em\u003e(peanutbase), Huayu 23 CK (wild type), Huayu 23-7-1 and Huayu 23-7-2\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-1096211/v2/c76941da66d1be32439adc97.png"},{"id":30219285,"identity":"abb82c36-7100-4a60-8ccc-7019f5abece8","added_by":"auto","created_at":"2022-12-12 17:55:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":55963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ebar \u003c/em\u003egene amplification products resolved on a 2% agarose gel\u003c/p\u003e\n\u003cp\u003eM: DL500 DNA Marker (TaKaRa, Dalian), PC: positive control (Cas9 empty vector), NC: negative control (Huayu 23), 1: HY23-7-1, 2: HY23-7-2\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-1096211/v2/b4d8f6cb00ac07835942dc8c.png"},{"id":30219284,"identity":"69c1f4e3-b1c5-4dd2-b4f1-1ae77d03064b","added_by":"auto","created_at":"2022-12-12 17:55:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1544987,"visible":true,"origin":"","legend":"\u003cp\u003eT1 plant set seeds, showing plant (a), pods (b) and T2 seeds (c)\u003c/p\u003e\n\u003cp\u003eWild type Huayu 23 (left), Huayu 23-7-1 (right)\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-1096211/v2/b537d45b06cccc83b5faf53d.png"},{"id":40057092,"identity":"4de7f3d2-e0e9-4fd9-a454-e4489664cf49","added_by":"auto","created_at":"2023-07-14 20:03:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4651774,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1096211/v2/686904c1-def6-43d5-b643-3805e48813b1.pdf"}],"financialInterests":"","formattedTitle":"In planta genetic transformation to produce CRISPRed high-oleic peanut","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRich in culinary oil and highly digestible protein, the cultivated peanut (\u003cem\u003eArachis hypogaea\u003c/em\u003e L.) occupies an important position in human and animal nutrition(Li et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Fatty acid profile is an indicator of its quality. Oleic and linoleic acids together constitute about 80% of total fatty acids in peanut seeds. As compared to linoleic acid, oleic acid is less prone to oxidation. Increase in oleic acid and decrease in linoleic acid in peanut seeds may result in extended shelf life of peanut produce and much more health benefits (Nkuna et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Zhao et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the cultivated peanut, \u003cem\u003eFAD2A\u003c/em\u003e and \u003cem\u003eFAD2B\u003c/em\u003e control the conversion of oleic acid to linoleic acid, and expression of the high-oleic phenotype (at least 70% oleic acid content) in peanut cultigen requires inactivation of both genes (Nawade et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Natural, chemical, and physical peanut mutants with high oleate have been reported and used in hybridization and backcross to develop high-oleic peanut cultivars (Wang et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)(Wang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)(Han et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In contrast to the lengthy process of conventional breeding, the speed of genome editing may be much faster. Using peanut germs with a cotyledon attached for transformation, Wen et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) demonstrated that TALENs-mediated targeted mutagenesis of \u003cem\u003eFAD2\u003c/em\u003e in peanut cv Yueyou 7 raised oleic acid content from 43\u0026ndash;60%~80%, while decreased linoleic acid content from 35.5% to lower than 20% (Wen et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Yuan et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) induced \u003cem\u003eFAD2B\u003c/em\u003e mutations in peanut protoplasts and hairy roots using CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 based genome editing. Zhang et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) bombarded the embryonic calli of peanut cv Luhua 11 with a CRISPR/Cas9 gene editing vector targeting \u003cem\u003eFAD2\u003c/em\u003e, and regenerated plants were obtained, but the fatty acid profiles of the descendants were not reported (Zhang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Tang et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) produced gene edit Huayu 23 lines with decreased saturated fatty acid and increased unsaturated fatty acid through knockout of \u003cem\u003eFATB\u003c/em\u003e (\u003cem\u003eAcyl-acyl carrier protein thioesterase B\u003c/em\u003e) by CRISPR/Cas9 system.\u003c/p\u003e \u003cp\u003eSuitable transformation procedures may facilitate the application of genome editing tools. Previously, node injection method, an easy-to-follow \u003cem\u003ein planta\u003c/em\u003e peanut transformation protocol, was developed at our laboratory (Wang et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). It was used to transfer soybean \u003cem\u003eSCTF-1\u003c/em\u003e gene to chill susceptible peanut, RT-PCR and Southern blot analysis confirmed the transgenic events. Peanut plants with \u003cem\u003eSCTF-1\u003c/em\u003e gene showed good tolerance to chill stress (V. M. Vacu, unpublished data). Likewise, transfer of \u003cem\u003eAhCYP\u003c/em\u003e, a bacterial wilt infection responsive gene (Ding et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) to Huayu 40, a susceptible peanut cultivar, enhanced bacterial wilt resistance of the recipient (吴琪 et al. 2019). This study aimed to test its effectiveness in inducing high-oleic peanut mutations using CRISPR/Cas9 technology.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material for genome editing and its cultivation\u003c/h2\u003e \u003cp\u003eHuayu 23, a popular peanut normal-oleic cultivar of runner market type widely accepted by growers and food processors in China, was used in this study. As expected, Sanger sequencing of its \u003cem\u003eFAD2A\u003c/em\u003e and \u003cem\u003eFAD2B\u003c/em\u003e and subsequent sequence alignment revealed that this cultivar had a mutated \u003cem\u003eFAD2A\u003c/em\u003e (448 G\u0026thinsp;\u0026gt;\u0026thinsp;A) and a wild type \u003cem\u003eFAD2B\u003c/em\u003e. Peanut for genome editing was sown under polyethylene film mulching in an isolated region in SPRI Laixi Experimental Station on May 5, 2021. Agronomic practices were followed as routine.\u003c/p\u003e \u003cp\u003e \u003cb\u003eVector construction and transformation of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eEscherichia coli\u003c/span\u003e\u003c/p\u003e \u003cp\u003eTarget site of sgRNA, 501\u0026ndash;520 position of the coding sequence (5\u0026rsquo;-catgaacaatccaccaggga-3\u0026rsquo;) of wild type \u003cem\u003eFAD2B\u003c/em\u003e, was selected using CRISPR-GE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://skl.scau.edu.cn/\u003c/span\u003e\u003cspan address=\"http://skl.scau.edu.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To facilitate ligation in genome editing vector construction, two oligos which would produce overhangs after mixing, denaturation and annealing were generated with online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://biogle.cn/index/excrispr\u003c/span\u003e\u003cspan address=\"http://biogle.cn/index/excrispr\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and synthesized (Tsingke, Qingdao) (FAD2B-1F: 5\u0026rsquo;-gggttgcatgaacaatccaccaggga-3\u0026rsquo;, FAD2B-1R: 5\u0026rsquo;-aaactccctggtggattgttcatgca-3\u0026rsquo;). CRISPR/Cas9 editing vector with target site incorporated was made with BGK41-Cas9 (Biogle Biotechnology Co. Ltd, Hangzhou) following manufacturer\u0026rsquo;s instructions. Briefly, BGK41-Cas9, oligo dimers and enzyme mix (Biogle CRISPR/Cas vector construction kit) were mixed on ice bath, and then incubated at 20 ℃ for 1 h. The ligation products were transformed into competent cells of \u003cem\u003eE. coli\u003c/em\u003e strain DH5α. Positive clones identified using colony-PCR with Cas9-F/Cas9-R primer pair (5\u0026rsquo;-tcgtgctgaccctgacactgtttga-3\u0026rsquo;, 5\u0026rsquo;- cttggcggtagccttgccgatttcc-3\u0026rsquo;) were sequenced with primer CXYW1 (5\u0026rsquo;- cccagtcacgacgttgtaaa-3\u0026rsquo;) (Tsingke, Qingdao) to confirm the inclusion of the target site in the vector. The newly constructed plasmid was named as BGK41-Cas9 recombinant vector FAD2B-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTransformation of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eAgrobacterium\u003c/span\u003e \u003cb\u003eand node injection transformation of peanut\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGenome editing construct was transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101 chemically competent cells (Veidi Biotech, Shanghai) according to the attached user\u0026rsquo;s guide. Preparation of \u003cem\u003eAgrobacterium\u003c/em\u003e for injection was based on Pan et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) with some modifications. Positive single clones were verified by bacterial suspension PCR using Cas9-F/Cas9-R primer pair. 100 \u0026micro;l of freshly prepared bacterial suspension were cultured in 10 ml of YEB liquid medium at 28 ℃ with agitation (250 rpm) until OD\u003csub\u003e600\u003c/sub\u003e reached 0.6\u0026ndash;0.8 (about 12 h). The cultures were centrifuged at 6 000 rpm for 1 min to collect the bacterial cells. Equal volume of infection solution containing 100 \u0026micro;mol/L acetosyringone (BBI Lifesciences, Hongkong), 10 mmol/L MES (Sangon Biotech, Shanghai) and 10 mmol/L MgCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO was added to the pellets. Resuspended bacterial pellets were used for injection. Node injection procedure was essentially the same as that in our previous report except for the plant age (Wang et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Injection was done between 6:00\u0026ndash;8:00 a.m. on July 17, 2021, and the positions injected were marked with threads (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Inverted U-shaped mental wires were used to facilitate the entry of pegs into the soil from higher nodes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFatty Acid Analysis Of Resultant T0 Seeds\u003c/h3\u003e\n\u003cp\u003ePods were harvested when matured (Sept. 17, 2021). These pods were sun-dried and hand shelled. Oleic and linoleic acid contents of the individual single seeds were predicted with NIRS (Wang et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Seeds with at least 74% oleic acid along with the untreated control Huayu 23 were further analyzed for fatty acids by gas-chromatography using cotyledonary slices follow the protocol of Yang et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eComparison of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eFAD2\u003c/span\u003e \u003cb\u003esequences between the high-oleic T0 seeds and Huayu 23\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eFAD2A\u003c/em\u003e and \u003cem\u003eFAD2B\u003c/em\u003e sequences of the high-oleic T0 seeds and Huayu 23 were amplified by PCR using primers aF19 (5\u0026rsquo;-gattactgattattgactt-3\u0026rsquo;)/R1 (5\u0026rsquo;- ctctgactatgcatcag-3\u0026rsquo;) and bF19 (5\u0026rsquo;-cagaaccattagctttg-3\u0026rsquo;)/R1, respectively (Patel et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), and DNA templates prepared from cotyledonary slices (Yu et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). PCR products were directly sequenced by Tsingke, Qingdao. Sequence comparison was done with the DNAStar Lasergene version 7.1.0.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAmplification of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ebar\u003c/span\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePCR amplification of \u003cem\u003ebar\u003c/em\u003e gene were performed using DNA templates prepared from Cas9 empty vector (positive control), Huayu 23 (negative control) and 2 high-oleic T0 seeds respectively, with Bar-7F and Bar-6 primer (Bar-7F: 5\u0026rsquo;-caccatcgtcaaccactaca-3\u0026rsquo;,bar-6r༚5\u0026rsquo;-acttcagcaggtgggtgta-3\u0026rsquo;). The PCR mixture (10 \u0026micro;1) consisted of 6.25 \u0026micro;1 of 2\u0026times;\u003cem\u003eTaq\u003c/em\u003e Plus Master Mix Ⅱ (Vazyme, Najing), 1 \u0026micro;1 of DNA template, 0.5 \u0026micro;1of primers (10 \u0026micro;M) each, and 4.25 \u0026micro;1 of double distilled water. PCR thermal profile was 95 ℃ 5 min, followed by 30 cycles of 95 ℃ for 40 s, 61 ℃ for 40 s, and 72 ℃ for 30 s, and a final extension of 72 ℃ for 10 min.\u003c/p\u003e\n\u003ch3\u003eCultivation Of T1 Plants\u003c/h3\u003e\n\u003cp\u003eTo obtain descendants as soon as possible, one T0 seed was sown in a pot in the winter of 2021, but unfortunately it died and did not set any pods. The remaining T0 seed along with a Huayu 23 seed was sown under film mulch on May 23, 2022. Both grew to maturity and were harvested on September 20, 2022.\u003c/p\u003e\n\u003ch3\u003eFatty Acid Profiling Of The T2 Seeds Harvested From T1 Plant\u003c/h3\u003e\n\u003cp\u003eOleic and linoleic acid contents of the T2 seeds from the T1 plant both as bulk seed sample and as individual single seeds were determined by NIRS (Wang et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Wang et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Huayu 23 CK was also analyzed for main fatty acid content.\u003c/p\u003e"},{"header":"Results And Analysis","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuality of the resultant T0 seeds and wild type Huayu 23 predicted by NIRS\u003c/h2\u003e \u003cp\u003eA total of 16 nodes were injected, which only resulted in 4 seeds. Among them, two seeds, Huayu 23-7-1 and Huayu 23-7-2, were classified as high-oleic by NIRS (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChemical quality of single T0 peanut seeds and Huayu 23 predicted by near infra-red spectroscopy\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIdentity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeneration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOleic acid (O) (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLinoleic acid (L) (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eO/L\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuayu 23 (CK)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuayu 23-7-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e74.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuayu 23-7-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e78.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMain fatty acids of the resultant seeds and wild type Huayu 23 by wet chemistry method\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFatty acid composition of the seeds of concern determined by GC was shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. There were drastic changes in oleic, linoleic and palmitic acid contents in Huayu 23-7-1 and Huayu 23-7-2. Oleic acid increased from 52.15% in Huayu 23 to over 80% in Huayu 23-7-1 and Huayu 23-7-2, linoleic acid dropped from 25.72% in Huayu 23 to lower than 1.70% in Huayu 23-7-1 and Huayu 23-7-2. Accordingly, the oleic acid to linoleic acid ratio (O/L) rose from around 2.03 to more than 47. Palmitic acid content in Huayu 23-7-1 and Huayu 23-7-2 was 8.07% and 7.39%, respectively, much lower than that in Huayu 23 (15.55%).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFatty acids (%) in single peanut seeds determined by gas chromatography\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIdentity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeneration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOleic acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLinoleic acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePalmitic acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO/L\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuayu 23 (CK)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuayu 23-7-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e47.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuayu 23-7-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e55.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eIn each column of the fatty acid content, figures followed by the same letter were not significantly different at 0.01 level. Fatty acid content was expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eFAD2A/FAD2B\u003c/span\u003e \u003cb\u003eGenotyping of the resultant T0 seeds and wild type Huayu 23\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMultiple alignment of \u003cem\u003eFAD2A/FAD2B\u003c/em\u003e sequences of different sources revealed that\u003c/p\u003e \u003cp\u003eHuayu 23, Huayu 23-7-1 and Huayu 23-7-2, all had a mutated \u003cem\u003eFAD2A\u003c/em\u003e (448 G\u0026thinsp;\u0026gt;\u0026thinsp;A), Huayu 23 possessed wild type \u003cem\u003eFAD2B\u003c/em\u003e, whereas both the high-oleic T0 mutants seeds had a mutant type \u003cem\u003eFAD2B\u003c/em\u003e (442A insertion) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The causal relationship between 442A insertion in \u003cem\u003eFAD2B\u003c/em\u003e and dysfunctional \u003cem\u003eFAD2B\u003c/em\u003e has been well clarified (Yu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). However, the 442A \u003cem\u003eFAD2B\u003c/em\u003e mutation was out of the scope of the anticipated targeting site, 501\u0026ndash;520 position of the coding sequence of wild type \u003cem\u003eFAD2B\u003c/em\u003e, where no mutation was detected.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAmplification of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ebar\u003c/span\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs expected, the 2 high-oleic peanut seeds produced a band with size equal to the positive control Cas9 empty vector, whereas the untreated Huayu 23 (negative control) yielded no band (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), verified that the 2 high-oleic peanut seeds were transformants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eT1 Plants Grown From High-oleic T0 Seeds\u003c/h3\u003e\n\u003cp\u003eThe two high-oleic T0 seeds along with the untransformed control were sown. Both T0 seeds developed into plants, but one died prior to flowering (Huayu 23-7-2). The remaining T1 plant (Huayu 23-7-1) grew normally and set a total of 73 seeds.\u003c/p\u003e\n\u003ch3\u003eOleic And Linoleic Acid Contents Of T1 Plant\u003c/h3\u003e\n\u003cp\u003eAll the seeds from the T1 plant Huayu 23-7-1 were firstly used as bulk seed sample in NIRS. The T1 plant had an oleic acid content of 79.07%, as against 44.52% in Huayu 23. Then sixty-eight well-developed T2 seeds were analyzed with NIRS for individual single seeds, and all were found to be high-oleic (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Likewise, forty-five Huayu 23 seeds were also used as individual single seed samples in NIRS analysis and, as expected, all were normal-oleic (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Compared to Huayu 23, the T1 plant was shorter and set slightly bigger pods (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Pod and seed weight of the T1 plant was also much higher (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Since the T1 plant and its parent were cultivated apart to avoid possible mechanical and biological mixing, it is still unknown these were caused by environmental factors or not.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChemical quality of bulk and single T2 peanut seeds and Huayu 23 predicted by NIRS\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIdentity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeneration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBulk/Single\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOleic acid (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLinoleic acid (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eO/L\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuayu 23 (CK)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBulk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e44.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuayu 23-7-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBulk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e79.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e15.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuayu 23 (CK)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSingle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e37.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e37.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuayu 23-7-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSingle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e75.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e22.91\u0026thinsp;\u0026plusmn;\u0026thinsp;7.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eFatty acid content and O/L were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE, where appropriate.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMain agronomic characters of gene-edited Huayu 23 T1 plant and Huayu 23\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlant identity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMain stem height (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLength of cotyledonary branches (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStem thickness (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRang of pod-bearing branches (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNumber of branches\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNumber of effective branches\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNumber of pods\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePod weight (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSeed weight (g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuayu 23 (CK)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e44.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e27.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuayu 23-7-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e61.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e36.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIt seems that biallelic genome editing in this report is questionable. In fact, in a separate study, using a novel technology and with the help of NIRS for bulk seed samples, we were able to obtain high-oleic peanut M1 chemical mutant single plants from a popular normal-oleic Spanish market type cultivar with wild type \u003cem\u003eFAD2A\u003c/em\u003e and wild type \u003cem\u003eFAD2B\u003c/em\u003e. In other words, it appeared that in these high-oleic mutants, not only were both \u003cem\u003eFAD2A\u003c/em\u003e and \u003cem\u003eFAD2B\u003c/em\u003e mutated, but that these mutations were homozygous (C. T. Wang, unpublished data). This may be ascribed to targeting reproductive cells at earlier stages rather than postzygotic tissues at later stages. The possibility that the mutant \u003cem\u003eFAD2B\u003c/em\u003e allele was in the background genotype of the cultivar used for transformation can be fully excluded. To be on the safe side, special care was taken to use breeder seeds, and the olei acid phenotype and \u003cem\u003eFAD2A/FAD2B\u003c/em\u003e genotype of Huayu 23 used for transformation were ascertained prior to the experiment.\u003c/p\u003e \u003cp\u003eIn this study, evidence from fatty acid profile of the T0 and T2 seeds, \u003cem\u003eFAD2B\u003c/em\u003e genotyping and \u003cem\u003ebar\u003c/em\u003e gene amplification all supported that genome editing of Huayu 23 was successful. Two peanut mutant T0 seeds with over 80% oleic acid were generated via CRISPR/Cas9 genome editing technology following the node injection method developed by Wang et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) (Wang et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and the high-oleic phenotype was expressed in T2 seeds clearly verified the usefulness of the peanut transformation protocol.\u003c/p\u003e \u003cp\u003eThe rationale behind the node injection method is that most of the peanut seeds set on the first (cotyledonary branches) and second pairs of branches, the possibility of harvesting sound mature kernels from the lower nodes was high, and that peanut cells that will develop into reproductive cells, or \u0026ldquo;primordial\u0026rdquo; reproductive cells, can be transformed (Wang et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Generally, only the first and second nodes counting from the intersection of the main stem and cotyledonary branches were injected at 30 days after sowing (Wang et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, in this study, when everything was ready, it was too late (63 days after sowing), only higher nodes could be injected. That is the reason why only a small number of seeds were harvested. Failing to edit in the anticipated target site may be due to the small population and/or the unsuitable oligos designed for genome editing vector construction, as the website for oligo design had no peanut genome option. Anyway, two CRISPRed high-oleic peanut seeds were identified from the 4 resultant seeds. Changes in oleic and linoleic acid contents in T0 and T2 seeds and the \u003cem\u003eFAD2B\u003c/em\u003e sequences (442A) of the two peanut T0 transformants demonstrated that the high-oleic phenotype was inheritable. In addition to high-oleic acid phenotype, high and stable productivity is a prerequisite for a peanut cultivar to be accepted by growers. It is still necessary to evaluate the overall performance of the derived lines in due course if commercialization is considered.\u003c/p\u003e \u003cp\u003eIn peanut, pods develop from pegs. One or multiple peg(s) is/are born at each node with peg(s), depending on cultivar (Nigam et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). In the case of multiple pegs, one injection may result in more than one pods, and in the meantime, the \u0026ldquo;primordial\u0026rdquo; reproductive cells at different developmental stages may increase the chances of being transformed. In this regard, node injection method is advantageous over flower injection. Our earlier study indicated that the method was genotype independent (Wang et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), where no tissue culture procedure was needed, expanding its scope of use. Nevertheless, we believe that in-depth developmental studies may help optimize peanut transformation efficiency of this method.\u003c/p\u003e \u003cp\u003eSince the peanut node injection transformation method is easy to implement, and as peanut is an oilseed crop and its seeds can be eaten raw, the node injection method may facilitate peanut molecular pharming. It is anticipated that the method, coupled with genome editing technology where necessary, will find wide utility in areas such as functional analysis of candidate peanut genes and development of genome edited peanut cultivars with improved safety quality, ideotype, and high and stable productivity. This node injection transformation method is not only useful to peanut, but also of some reference to other seed plant species.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eTo sum up, using the node injection transformation method and a CRISPR/Cas9 construct targeting \u003cem\u003eFAD2B\u003c/em\u003e, two high-oleic peanut mutant seeds with 442A insertion in \u003cem\u003eFAD2B\u003c/em\u003e were generated from normal-oleic cultivar Huayu 23 already having dysfunctional \u003cem\u003eFAD2A\u003c/em\u003e. Amplification of \u003cem\u003ebar\u003c/em\u003e gene verified that the 2 high-oleic peanut seeds were true transformants. One of the T0 seed developed into a healthy plant. NIRS analysis demonstrated that the high-oleic phenotype was expressed in T2 seed generation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eFAD2A\u003c/em\u003e\u0026nbsp; \u003cem\u003efatty acid desaturase\u003c/em\u003e \u003cem\u003e2A\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFAD2B\u003c/em\u003e\u0026nbsp; \u0026nbsp;\u003cem\u003efatty acid desaturase\u003c/em\u003e \u003cem\u003e2B\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGC \u0026nbsp; \u0026nbsp; gas-chromatography\u003c/p\u003e\n\u003cp\u003eMES \u0026nbsp; \u0026nbsp;2-(N-morpholino) ethanesulfonic acid sodium salt\u003c/p\u003e\n\u003cp\u003eNIRS \u0026nbsp; near infra-red spectroscopy\u003c/p\u003e\n\u003cp\u003eOD \u0026nbsp; \u0026nbsp; optical density\u003c/p\u003e\n\u003cp\u003eTALENs transcription activator-like effector nucleases\u003c/p\u003e\n\u003cp\u003eYEB \u0026nbsp; \u0026nbsp;yeast extract broth\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies involving human participants or animals performed by any of the authors. This study was approved by the ethical committee of Shandong Peanut Research Institute. Informed consent was obtained from all the authors of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e All of the authors of \u0026ldquo;\u003cem\u003eIn planta\u003c/em\u003e genetic transformation to produce CRISPRed high-oleic peanut\u0026rdquo; here declare that there is no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCTW designed and supervised the experiment. HWH and ZWW conducted the transformation experiment. ZY, CJJ, XZW and XSS were responsible for peanut cultivation, NIR analysis, and regent supplies. STY performed GC analysis. CTW, HHW, and STY prepared the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe express our sincere thanks to the financial support from Taishan Industry Leading Talents Special Fund (LJNY201808), Guangdong Program for Science \u0026amp; Technology Plan (2020B020219003), China Agricultural Research System (CARS-13), and Agricultural Science \u0026amp; Technology Innovation Project of Shandong Academy of Agricultural Sciences (CXGC2022A03).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDing YF, Wang CT, Tang YY et al (2012) Isolation and analysis of differentially expressed genes from peanut in response to challenge with Ralstonia solanacearum. 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ZL 2019 1 0820261.0\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-growth-regulation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"grow","sideBox":"Learn more about [Plant Growth Regulation](https://www.springer.com/journal/10725)","snPcode":"10725","submissionUrl":"https://submission.nature.com/new-submission/10725/3","title":"Plant Growth Regulation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Groundnut, Arachis, Genome editing, High oleate, bar","lastPublishedDoi":"10.21203/rs.3.rs-1096211/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1096211/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn contrast to its normal-oleic counterpart, high-oleic peanut has better keeping quality and multiple health benefits. Breeding high-oleic peanut through conventional means is a tedious process generally requiring several years. Genome editing, however, may shorten the duration. In this study, node injection method was used to transform normal-oleic Huayu 23, a popular peanut cultivar having dysfunctional \u003cem\u003eFAD2A\u003c/em\u003e and functional \u003cem\u003eFAD2B\u003c/em\u003e, with CRISPR/Cas9 construct targeting \u003cem\u003eFAD2B\u003c/em\u003e, and two T0 seeds with over 80% oleic acid and 442A insertion in \u003cem\u003eFAD2B\u003c/em\u003e were obtained. A T1 plant grown from the viable T0 seed produced high-oleic seeds. As a genotype-independent, simple and easy method for peanut genetic transformation, node injection has great potential in functional analysis of genes and peanut varietal improvement.\u003c/p\u003e","manuscriptTitle":"In planta genetic transformation to produce CRISPRed high-oleic peanut","msid":"","msnumber":"","nonDraftVersions":[{"code":"","date":"2023-06-03 19:18:37","doi":"","editorialEvents":[{"type":"reviewerAgreed","content":"","date":"2023-06-03T19:18:37+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-06-02T02:56:46+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant Growth Regulation","date":"2023-05-30T07:31:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Growth Regulation","date":"2023-05-26T11:23:06+00:00","index":"","fulltext":""},{"type":"decision","content":"Major revisions","date":"2023-05-10T23:04:24+00:00","index":"","fulltext":""}],"status":"private","journal":{"display":true,"email":"[email protected]","identity":"plant-growth-regulation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"grow","sideBox":"Learn more about [Plant Growth Regulation](https://www.springer.com/journal/10725)","snPcode":"10725","submissionUrl":"https://submission.nature.com/new-submission/10725/3","title":"Plant Growth Regulation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}},{"code":2,"date":"2022-12-12 17:55:32","doi":"10.21203/rs.3.rs-1096211/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2022-12-03T10:51:48+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-10-30T07:53:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant Growth Regulation","date":"2022-10-21T01:24:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-10-20T08:08:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Growth Regulation","date":"2022-10-18T12:38:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-growth-regulation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"grow","sideBox":"Learn more about [Plant Growth Regulation](https://www.springer.com/journal/10725)","snPcode":"10725","submissionUrl":"https://submission.nature.com/new-submission/10725/3","title":"Plant Growth Regulation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f779434a-b2e6-40d9-84c1-80c5a9d02fd9","owner":[],"postedDate":"December 12th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-07-14T20:03:45+00:00","versionOfRecord":{"articleIdentity":"rs-1096211","link":"https://doi.org/10.1007/s10725-023-01031-y","journal":{"identity":"plant-growth-regulation","isVorOnly":false,"title":"Plant Growth Regulation"},"publishedOn":"2023-06-26 00:00:00","publishedOnDateReadable":"June 26th, 2023"},"versionCreatedAt":"2022-12-12 17:55:32","video":"","vorDoi":"10.1007/s10725-023-01031-y","vorDoiUrl":"https://doi.org/10.1007/s10725-023-01031-y","workflowStages":[]},"version":"v2","identity":"rs-1096211","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1096211","identity":"rs-1096211","version":["v2"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}