Coupling FT-Mediated Speed Breeding and CRISPR/Cas9 for Rapid Trait Improvement in Nicotiana tabacum

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M. Nguyen, Ha C. Chu, Oleg S. Nikonov, Ekaterina Y. Nikonova, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8772804/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract The commercial deployment of genome-edited crops is frequently bottlenecked by the extended juvenile phases of perennial species and the complex regulatory landscapes governing plants with integrated exogenous DNA. Speed breeding protocols, which utilize environmental manipulation to accelerate development, have proven effective for annual cereals but often lack efficacy in perennials. In this study, we report a genetic speed breeding system that couples the overexpression of the floral integrator FLOWERING LOCUS T ( FT ) with CRISPR/Cas9-mediated genome editing. Using Nicotiana tabacum as a model for polyploid crops, we demonstrate that constitutive expression of Arabidopsis thaliana FT ( AtFT ) reduces the generation time from 12 weeks to approximately 3 weeks without compromising fertility or seed viability. To leverage this acceleration for trait improvement, we engineered a binary vector co-expressing AtFT and a CRISPR/Cas9 cassette targeting the NtDFR ( dihydroflavonol 4-reductase ) loci. This integrated system induced rapid flowering and simultaneous disruption of anthocyanin biosynthesis, yielding ntdfr mutants with a distinct white-flower phenotype. Importantly, the edited alleles were heritable, while the FT-Cas9 transgene could be segregated out in the next generation, restoring the wild-type photoperiodic response in the edited progeny. This transgenic facilitator approach offers a scalable platform for the rapid introgression of edited traits into recalcitrant crop species, potentially reducing the breeding cycle of perennials from years to months. Speed breeding CRISPR/Cas9 FLOWERING LOCUS T flower color DFR Figures Figure 1 Figure 2 Introduction The acceleration of crop improvement cycles is a fundamental objective of modern agriculture, driven by the urgent need to adapt global food systems to a rapidly changing climate and expanding population. While recent advances in genomic technologies have provided the tools to identify and engineer beneficial alleles, the rate of genetic gain is ultimately limited by the biological generation time of the target species (Chen et al. 2019 ). This limitation is particularly acute in perennial crops and woody species, where the juvenile can span from several years to over a decade (Flachowsky et al. 2009 ; Prado et al. 2024 ). Consequently, the introgression of a single edited trait into a commercial perennial cultivar via conventional backcrossing can be prohibitively slow and resource-intensive. Although genome editing technologies, particularly CRISPR/Cas systems, have revolutionized trait development (Tuncel et al. 2023 ; Tuncel et al. 2025 ) their application in perennials remains constrained by the time required to generate mature, edited plants.To address this, "speed breeding" methodologies manipulating photoperiod and temperature were developed (Watson et al. 2018 ). However, environmental speed breeding is often ineffective for species with complex dormancy requirements or obligate short-day responses. For such recalcitrant species, genetic intervention using the FLOWERING LOCUS T ( FT ) gene offers a potent alternative. FT encodes florigen, a conserved mobile protein that initiates floral development (Corbesier and Coupland 2005 ; Wickland and Hanzawa 2015 ). Overexpression of FT induces precocious flowering in diverse genera, including Populus , Malus , and Citrus Citrus (Böhlenius et al. 2006 ; Klocko et al. 2016 ; Adeyemo et al. 2017 ; Khan et al. 2020 ) Despite its utility, constitutive FT overexpression in a final crop product is undesirable due to potential pleiotropic effects on yield and architecture. Furthermore, the persistence of the transgene triggers stringent regulatory oversight (Strauss et al. 2015 ; Buchholzer and Frommer 2023 ). Therefore, a unified system that utilizes FT to accelerate the breeding cycle during editing, but allows for subsequent transgene removal, would combine the speed of genetic induction with the regulatory advantages of transgene-free editing. Here, we establish such a system in Nicotiana tabacum , utilizing an integrated AtFT overexpression and CRISPR/Cas9 module to achieve rapid generation cycling and efficient editing of the NtDFR gene family. Materials and methods Plant Materials and Growth Conditions Arabidopsis thaliana (ecotype Col-0) and tobacco ( Nicotiana tabacum cv. K326) were used in this study. All plants were cultivated in a controlled environment growth chamber maintained under a 16:8 h light/dark photoperiod at 24–28°C. Vector constructions The full-length coding sequence (CDS) of AtFT (AT1G65480) was amplified from Arabidopsis thaliana floral bud tissues using specific primers (Table S1 ) and Q5 high-fidelity DNA polymerase (M0491L, NEB). The 528 bp amplicon was cloned into the pMDC43 binary vector (Curtis and Grossniklaus 2003 ) between the Kpn I and Sac I restriction sites, placing it under the CaMV 35S promoter to generate pMDC-AtFT. The plasmid was confirmed by Sanger sequencing. To create the integrated editing vector, the CaMV35S::AtFT::Nos cassette was amplified from pMDC-AtFT and subcloned into the pFGC-Cas9-GW backbone (Nguyen et al. 2021 ) at the Spe I site via Gibson Assembly NEBuilder® HiFi DNA Assembly Cloning Kit (E5520S, NEB ) (Gibson et al. 2009 ). The plasmid was confirmed by Sanger sequencing. The entry vector, pAtU6-GW, driven by the AtU6 promoter and flanked by attL1-attL2 sequence was synthesized by Twist Bioscience (CA, USA). For gRNA design, the web tool, CCTop (Stemmer et al. 2015 ) was used to design. A 23 nucleotides sequence that is identical in NtDFR1 (Genebank: EF421429) and NtDRF2 (Genebank: EF421430) genes at the first exon of both two genes was selected. DNA oligonucleotide pairs (Table S1 ) was synthesized by PhusaGenomics (Can Tho, Vietnam) and annealed to generate dimer. The dimer was cloned into BbsI sites of pAtU6-GW to create pAtU6-NtDFR, which was then mobilized into the binary vector, pFGC-Cas9-AtFT-GW, at the attR1-attR2 site using Gateway™ LR Clonase (Invitrogen) to create pFGC-Cas9-AtFT-NtDRF. Agrobacterium -mediated transformation The binary vectors were electroporated into Agrobacterium tumefaciens strain GV3101. Tobacco leaf disk transformation was performed as described by (Horsch et al. 1985 ) with modifications. Leaf explants were infected with bacterial suspension (OD 600 =0.5) for 30 minutes and co-cultivated for 48 hours in the dark. Explants were subsequently selected on Murashige and Skoog (MS) medium supplemented with 500 mg/L cefotaxime and either 20 mg/L hygromycin (for pMDC-AtFT) or 3 mg/L glufosinate (for pFGC-Cas9-AtFT-NtDFR). Molecular and Phenotypic Characterization Chromosomal DNA was isolated following routine isolation techniques from young leaf tissues of the overexpression or CRISPR/Cas9 transgenic lines. The PCR amplification was performed using gene-specific primers with Q5 High fidelity DNA polymerase (M0491L, NEB). Primers used for CRISPR genotyping were listed in Table S1 . PCR products were extracted and purified from the agarose gel using GeneJET Gel Extraction Kit (K0691, Thermo Scientific), subsequently cloned into pJET1.2 Cloning Vector (K1231, Thermo Scientific). Individual clones were sequenced by Sanger sequencing using the pJET1.2-F and pJET1.2-R primers. For CAPS assay, the PCR products were incubated with PvuII enzyme (R0151S, NEB) at 37 0 C overnight. After digestion, the mixtures were loaded in 2.5% agarose gel. Shoot apices from control and overexpression lines at 15 days after germination were collected. RNA was extracted using TRIzol reagent (Invitrogen), purified with FastPure Plant Total RNA Isolation Kit (Vazyme, China) and treated with TURBO™ DNase following the manufacturer's instructions. cDNA was synthesized using oligo(dT) primers (15-mer) and HiScript IV 1st Strand cDNA Synthesis Kit (Vazyme, China) following the manufacturer's instructions. Transcript levels of genes were determined by qRT-PCR using an QuantStudio 5 real-time PCR following the SYBR Green method (Applied Biosystems). Gene expression levels were normalized to the expression of the Actin housekeeping gene and analyzed with 2 −ΔΔ C T method (Livak and Schmittgen 2001 ). Primers used for qRT-PCR were listed in Supplemental Table S1 . Total anthocyanin content was determined according to the method described by Weiss and Halevy ( 1989 ). Briefly, corollas were snap-frozen in liquid nitrogen and stored at -80°C until analysis. A total of 100 mg of powdered samples was incubated in 600 µL of extraction buffer (methanol containing 1% HCl) in the dark for 24 hours at 4°C. The addition of 200 µL of water and 200 µL of chloroform was followed by centrifugation at 12,000 × g for 10 minutes at 4°C. Absorbance of the supernatant was measured at 530 nm (A530) and 657 nm (A657) using microplate reader. Total anthocyanin content was calculated based on the molar absorbance of cyanidin-3-O-glucoside, with A530–0.25 × A657 used for quantification. Each sample was extracted and measured in five biological independent experiments. Statistical Analysis Sample means between genotypes were compared using Student's t -test or one-way ANOVA followed by Tukey's HSD test. All statistical analyses were done using GraphPad Prism 9.0 (GraphPad Software, Inc.) Results and discussions Ectopic Expression of AtFT Accelerates Floral Transition in Tobacco To examine the effect of ectopic expression FT on growth and development of the plant, we analyzed tobacco lines transformed with the AtFT overexpression vector (pMDC-AtFT) (Fig. 1 A). Five independent transgenic events were generated and confirmed via PCR genotyping (Fig. 1 B). Important, the T0 transgenic lines produced flowers under in vitro conditions (Fig. S1 A). To evaluate the impact of FT on plant and growth of the transgenic lines, T1 plants from two independent overexpression lines (OX6 and OX8) were sown and observed. While wild-type (WT) K326 plants required approximately 12 weeks to initiate flowering under long-day conditions, AtFT -overexpressing lines (OX6 and OX8) produced visible floral buds as early as 3 weeks post-germination (Fig. 1 C), and set fruit within 3 weeks after germination (Fig. S1 B). This represents a four-fold acceleration of the generation cycle. Importantly, this rapid development did not compromise reproductive viability. The transgenic lines produced viable seeds (Fig. S1 C), a prerequisite for any breeding application. The plants exhibited a short stature solely due to the extreme reduction in the vegetative growth phase; however, germination rates and seedling vigor remained comparable to the wild type (Fig. S1 B, Table S2 ). To mechanistically validate that the observed phenotype was driven by the integration of the heterologous AtFT into the tobacco floral network, we quantified the expression of endogenous floral identity genes. NtSOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1) and NtAP1 (APETALA1) are key integrators acting downstream of the florigen signal (Lee and Lee 2010 ). Our qRT-PCR analysis revealed a significant upregulation of both NtSOC1 and NtAP1 in the shoot apices of transgenic plants at 12 days post-germination (Fig. 1 D). This molecular evidence confirms that AtFT acts as a potent floral activator in N. tabacum , effectively bypassing the juvenile phase requirements. Simultaneous Editing and speed breeding using the AtFT -CRISPR System Having validated the speed breeding component, we deployed the integrated vector pFGC-Cas9-AtFT-NtDFR (Fig. 2 A) to test the system's capacity for simultaneous rapid cycling and genome editing. The NtDFR genes were selected as a target because disruption of anthocyanin biosynthesis yields a readily scorable white-flower phenotype. To simultaneously mutate both DFR genes in tobacco, a guide RNA targeting the first exon of both genes was selected to construct the pFGC-Cas9- AtFT - NtDFR binary vector. Importantly, the single guide RNA (sgRNA) was designed to overlap a Pvu II restriction site, facilitating rapid genotypic screening of edited lines via Cleaved Amplified Polymorphic Sequences (CAPS) analysis. (Fig. 2 B). Agrobacterium -mediated transformation of the tobacco cultivar K326 generated four basta-resistant lines (CR7, CR30, CR31, and CR39) that carried AtFT and CRISPR/Cas9 components (Fig. S2 ). To examine the editing efficacy of Cas9 in the speed breeding complex, these four transgenic lines were subjected to genotyping using the PCR-CAPS assay with Pvu II. While the wild-type amplicon was completely digested by the Pvu II restriction enzyme, all four transgenic events showed resistance to Pvu II digestion in either NtDFR1 , NtDFR2 , or both (Fig. 2 C). The CAPS assay analysis suggested that the transgenic lines CR7 and CR39 were heterozygous for mutations in both NtDFR1 and NtDFR2 genes, whereas the edited lines CR30 and CR31 possessed heterozygous mutations only in the NtDFR1 gene (Fig. 2 C). Sanger sequencing was used to further characterize the mutants CR7 and CR30, confirming that mutant line CR7 possessed a 3-bp and 3-bp deletion for DFR1 and DFR2 respectively, while a 2-bp deletion was obtained in the edited line CR30 (Fig. 2 D). These results suggest that the gene editing efficacy of the Cas9 system in this accelerated cycle complex is as high as in the original binary backbone without AtFT (Do et al. 2019 ; Nguyen et al. 2021 ). We next examined the inheritance of the mutations in the T1 generation. Plants from the two edited lines, CR7 and CR30, were subjected to CAPS assays for genotyping. Gel electrophoresis results confirmed that the mutations identified in the T0 generation were successfully transmitted to the next generation (Fig. S3 ). Consistent with the genetic characteristics of the T0 progenitors (CR7: DFR1/dfr1 , DFR2/dfr2 ; CR30: DFR1/dfr1 , DFR2/DFR2 ), T1 progeny from the CR7 line segregated to produce single mutants for dfr1 and dfr2 , while only single dfr1 mutants were obtained from CR30. Notably, we were able to successfully recover homozygous double mutants ( dfr1 dfr2 ) in the T1 generation derived from CR7. As NtDFR is known to determine flower color in tobacco (Kazama et al. 2013 ; Lim et al. 2016 ), we analyzed floral pigmentation and quantified total anthocyanin content in the dfr mutant lines. While all plants carrying AtFT flowered early (Fig. 2 E), variations in flower color were strictly correlated with the presence of specific mutations (Fig. 2 F, Fig. S4 ). While dfr1 single mutants produced pale pink flowers, dfr2 single mutants produced white flowers (Fig. 2 E, Fig. S4 A). Anthocyanin levels extracted from corolla tissues of fully expanded flowers showed consistency with visible petal color intensity (Fig. 2 F, Fig. S4 B). The anthocyanin content of dfr1 single mutants was reduced by approximately half compared to wild-type plants, while dfr2 single mutants contained 90% and 72% less anthocyanin than WT and dfr1 single mutants, respectively. These results confirm that NtDFR1 and NtDFR2 are responsible for the color phenotype in tobacco flowers and suggest that NtDFR2 is the major contributor to the pink color, as has been reported in tobacco cv. Xanthi (Kazama et al. 2013 ). In summary, our study establishes a robust integrated speed-breeding system that couples the floral induction of AtFT with the precision of CRISPR/Cas9 editing. We demonstrated that this dual-function system can compress the breeding cycle of N. tabacum from months to weeks while achieving high-efficiency mutagenesis of the NtDFR loci. Crucially, the independent segregation of the editing machinery and the targeted mutation in the T1 generation allows for the rapid recovery of transgene-free, homozygous edited lines. This capability is particularly significant for overcoming the regulatory and biological barriers associated with the genetic improvement of woody perennials and crops with extended juvenile phases, providing a versatile toolkit for modern crop breeding. Declarations Conflict of Interest The authors declare no conflict of interest Funding This research was supported by the Vietnam Academy of Science and Technology, and the Russian Science Foundation in a bilateral project coded QTRU06.08/24–26 (or 24-44-04007). Author Contribution CXN and PTD conceived and conceptualized the study. CXN designed, performed the experiments, and analyzed data. AMN performed experiments. PTD managed the project. CXN wrote the first draft of the manuscript, which was subsequently revised and proofread by all authors. 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Germination rate UncropedFig.1BActin.jpg UncropedFig.2CNtDFR2.jpg UncropedFig.2CNtDFR1.jpg UncropedFig.1BAtFT.jpg UncropedFig.S2AtFT.jpg UncropedFig.S2Actin.jpg UncropedFig.S2Cas9.jpg UncropedFigS3.CR30NtDFR1.jpg UncropedFigS3.CR7NtDFR1.jpg UncropedFigS3.CR7NtDFR2.jpg UncropedFigS3.CR30NtDFR2.jpg Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 04 May, 2026 Reviews received at journal 07 Apr, 2026 Reviews received at journal 01 Apr, 2026 Reviewers agreed at journal 23 Mar, 2026 Reviewers agreed at journal 22 Mar, 2026 Reviewers invited by journal 21 Mar, 2026 Editor assigned by journal 09 Feb, 2026 Submission checks completed at journal 07 Feb, 2026 First submitted to journal 03 Feb, 2026 You are reading this latest preprint version 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-8772804","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":598765495,"identity":"e7962853-68ff-43bf-b62d-8676a7fc42bf","order_by":0,"name":"Anh T. M. 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Nguyen","email":"data:image/png;base64,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","orcid":"","institution":"Vietnam Academy of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Cuong","middleName":"X.","lastName":"Nguyen","suffix":""}],"badges":[],"createdAt":"2026-02-03 08:09:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8772804/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8772804/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103709744,"identity":"72497bf8-14ea-4e4b-9f85-44d6f4082f29","added_by":"auto","created_at":"2026-03-02 03:17:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":344911,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAtFT\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e reduces flowering time in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNicotiana tabacum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (A) Schematic representation of the binary vector construct designed for \u003cem\u003eAtFT\u003c/em\u003e overexpression under the control of the 35S promoter. (B) PCR-based genotyping of T0\u003csub\u003e \u003c/sub\u003etransgenic lines transformed with \u003cem\u003eAtFT\u003c/em\u003e. The upper panel displays PCR amplification products confirming the presence of the \u003cem\u003eAtFT\u003c/em\u003e transgene. Genomic DNA from wild-type \u003cem\u003eN. tabacum\u003c/em\u003e cv. K326 (WT) and five independent transgenic lines (lines 1-5) was used as template. \u003cem\u003eNtActin\u003c/em\u003e (lower panel) served as a positive PCR control for genomic DNA integrity. (C) Transcript levels of \u003cem\u003eNtSOC1\u003c/em\u003e and \u003cem\u003eNtAP1\u003c/em\u003e genes in the shoot apex of wild-type and two transgenic lines (OX6 and OX8) at 12 days post-germination. Expression levels were normalized to \u003cem\u003eNtActin\u003c/em\u003e transcript abundance. Data represent mean values ± standard error of the mean (SEM) from four biological replicates (n = 4). Numbers above bars indicate p-values from ANOVA with Tukey's HSD post-hoc test. (D) Representative images of wild-type and \u003cem\u003eAtFT\u003c/em\u003e-overexpressing lines (OX6 and OX8) 25 days post-germination. Transgenic lines exhibited early flowering, observed as early as 25 days post-germination. Scale bar = 3 cm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/f52a008b08b2a42a8ab8d5c3.png"},{"id":103709745,"identity":"3e3d0d45-9ab0-422b-8480-2ae1973596b1","added_by":"auto","created_at":"2026-03-02 03:17:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":360624,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntegration of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAtFT\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and CRISPR/Cas9-induced mutations in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNtDFR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes in tobacco.\u003cbr\u003e\n \u003c/strong\u003e(A) Schematic representation of the integrated pFGC-Cas9-AtFT-GW binary vector construct and the \u003cem\u003eAtU6\u003c/em\u003e entry construct. The \u003cem\u003eBbsI \u003c/em\u003erestriction site was used to assemble the spacer sequences. Abbreviations: BAR, \u003cem\u003eBasta\u003c/em\u003eselection gene; GmUBQ, soybean \u003cem\u003eUBQ\u003c/em\u003e constitutive promoter; hCAS9, human \u003cem\u003eCas9\u003c/em\u003e; Nos-Ter, \u003cem\u003eNopaline Synthase\u003c/em\u003e terminator; attR1-ccdB-attR2, Gateway recombination sites; LB, T-DNA left border; RB, T-DNA right border; pU6, \u003cem\u003eArabidopsis U6\u003c/em\u003e promoter; gRNA, guide RNA.(B) Schematic diagram showing the target sites in the first exon of \u003cem\u003eNtDFR1\u003c/em\u003e and \u003cem\u003eNtDFR2\u003c/em\u003e. The sgRNA sequence containing a \u003cem\u003ePvuII\u003c/em\u003e restriction site is underlined, and the PAM sequence is shown in blue. Red arrows indicate the translation start sites, and black arrows represent primers used for PCR to detect induced mutations. (C) CAPS-based (PCR/RFLP) genotyping of four T0 transgenic lines (CR7, CR30, CR31, and CR39) transformed with pFGC-Cas9-AtFT-NtDFR, along with wild-type K326 (WT) plants. PCR products were digested with \u003cem\u003ePvuII\u003c/em\u003e, which recognizes a site overlapping the CRISPR/Cas9 target. WT: untransformed control; \"−\": undigested; \"+\": digested with \u003cem\u003ePvuII\u003c/em\u003e. Red arrowheads indicate mutated bands (un-digested), and blue arrowheads denote wild-type fragments cleaved by \u003cem\u003ePvuII\u003c/em\u003e. (D) Sequence alignments of the \u003cem\u003eNtDFR\u003c/em\u003e target region in WT and representative mutant lines (CR7, CR30). Dashes indicate deleted nucleotides. The PAM sequence is highlighted in blue, and the \u003cem\u003ePvu\u003c/em\u003eII restricted sequence is underlined. Nucleotides different between DRF1 and DFR2 were orange colored. Allelic mutations are described to the right of the sequences. (E) Phenotypic comparison of flowering time and flower color in CR7 (\u003cem\u003eDFR1, DFR2\u003c/em\u003e) and the dfr2 mutant transgenic line CR7 (\u003cem\u003eDFR1, dfr2\u003c/em\u003e). (F) Quantification of total anthocyanin content in corolla tissues of WT and mutant lines. Data represent mean ± SD (n=3). Statistical analysis was done by one-way ANOVA followed by a post-hoc Tukey's multiple range test. Letters indicate significant differences at p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/2eedc7164a38f7d5b78e34d9.png"},{"id":104407618,"identity":"fed99a67-01c2-4c14-a1f1-c842fd2a8db0","added_by":"auto","created_at":"2026-03-11 12:39:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1321517,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/af278254-50b5-49bc-80ce-52873a3ef321.pdf"},{"id":103709759,"identity":"e445e63b-53b4-4bcb-ad93-69dd57cdc8eb","added_by":"auto","created_at":"2026-03-02 03:17:30","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10074724,"visible":true,"origin":"","legend":"","description":"","filename":"FigS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/a214951d23fa69fef3162c20.tif"},{"id":103709751,"identity":"2b63cf87-cf4a-4974-98e8-d3474d30b2b2","added_by":"auto","created_at":"2026-03-02 03:17:30","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5145834,"visible":true,"origin":"","legend":"","description":"","filename":"FigS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/c770fe53b8afbc237b8c3eee.tif"},{"id":103709752,"identity":"ddc4f053-b24e-4d2c-8183-d4aaac8abf4e","added_by":"auto","created_at":"2026-03-02 03:17:30","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4898162,"visible":true,"origin":"","legend":"","description":"","filename":"FigS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/2ac82baf37955cae02573a8c.tif"},{"id":104399683,"identity":"d70db41d-b4ba-4387-a0e5-024265218d4a","added_by":"auto","created_at":"2026-03-11 12:07:13","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":6205280,"visible":true,"origin":"","legend":"","description":"","filename":"FigS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/d6c46a6145678bb7ab760bad.tif"},{"id":103709746,"identity":"cacf6dd9-b011-45d0-8f23-ed43e5b58db0","added_by":"auto","created_at":"2026-03-02 03:17:30","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":13644,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementarysequences\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"supplementarysequences.docx","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/e9454664ba83686bb1114302.docx"},{"id":104399841,"identity":"a3c1961c-36d6-4a8d-be99-1f738bdf449d","added_by":"auto","created_at":"2026-03-11 12:07:49","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":12110,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1\u003c/strong\u003e. List of primers used in the study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S2\u003c/strong\u003e. Germination rate\u003c/p\u003e","description":"","filename":"Supplematarytables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/6b3de48edce5c412a64625f6.xlsx"},{"id":103709758,"identity":"5c4c199c-95ac-4353-82bb-8d6056c5d4e9","added_by":"auto","created_at":"2026-03-02 03:17:30","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":241633,"visible":true,"origin":"","legend":"","description":"","filename":"UncropedFig.1BActin.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/a625c396428f0042e25ad0d1.jpg"},{"id":103709761,"identity":"245f63d7-98e8-4a25-9388-d60d0e35129e","added_by":"auto","created_at":"2026-03-02 03:17:31","extension":"jpg","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":717688,"visible":true,"origin":"","legend":"","description":"","filename":"UncropedFig.2CNtDFR2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/8403c6ba355b2a29d2320317.jpg"},{"id":104399948,"identity":"bb654a98-df19-4833-823e-58594479b13c","added_by":"auto","created_at":"2026-03-11 12:08:16","extension":"jpg","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":478762,"visible":true,"origin":"","legend":"","description":"","filename":"UncropedFig.2CNtDFR1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/9cc1a28879d6ea2adf98b0a9.jpg"},{"id":103709753,"identity":"d76f233a-d10e-4db4-82af-f73149e38dec","added_by":"auto","created_at":"2026-03-02 03:17:30","extension":"jpg","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":702077,"visible":true,"origin":"","legend":"","description":"","filename":"UncropedFig.1BAtFT.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/b36e8cc3e195ee9271740652.jpg"},{"id":104400167,"identity":"3226343d-4f57-4bd8-a54b-545ce18d1ca2","added_by":"auto","created_at":"2026-03-11 12:09:05","extension":"jpg","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":471467,"visible":true,"origin":"","legend":"","description":"","filename":"UncropedFig.S2AtFT.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/b73a80d4e8c06cd6db6a103f.jpg"},{"id":103709747,"identity":"e41e9102-bdc6-4251-8432-88f04cb4536e","added_by":"auto","created_at":"2026-03-02 03:17:30","extension":"jpg","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":261716,"visible":true,"origin":"","legend":"","description":"","filename":"UncropedFig.S2Actin.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/991f7e7f10d0035bae5c8b90.jpg"},{"id":103709760,"identity":"bd1d3c3a-55dc-4c50-aca2-720b6cd5260e","added_by":"auto","created_at":"2026-03-02 03:17:30","extension":"jpg","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":286639,"visible":true,"origin":"","legend":"","description":"","filename":"UncropedFig.S2Cas9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/00a5dbfe2a8066dc726d7af8.jpg"},{"id":103709756,"identity":"385c7895-a1f2-4bdb-93f2-1173ad85a87c","added_by":"auto","created_at":"2026-03-02 03:17:30","extension":"jpg","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":1876273,"visible":true,"origin":"","legend":"","description":"","filename":"UncropedFigS3.CR30NtDFR1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/226e932aaeea4eef6b035ac9.jpg"},{"id":103709762,"identity":"fee95db9-a179-4cec-b16e-714f0079d660","added_by":"auto","created_at":"2026-03-02 03:17:31","extension":"jpg","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":1807031,"visible":true,"origin":"","legend":"","description":"","filename":"UncropedFigS3.CR7NtDFR1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/6342d8e158c802895e9cf5af.jpg"},{"id":103709755,"identity":"bae0c311-f0a4-42e6-a5f8-a7420f4f4b00","added_by":"auto","created_at":"2026-03-02 03:17:30","extension":"jpg","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":1534215,"visible":true,"origin":"","legend":"","description":"","filename":"UncropedFigS3.CR7NtDFR2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/573b4f756acfe9715c58f199.jpg"},{"id":104399976,"identity":"f73968a9-d994-48d9-ae20-d428d7725870","added_by":"auto","created_at":"2026-03-11 12:08:21","extension":"jpg","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":1654549,"visible":true,"origin":"","legend":"","description":"","filename":"UncropedFigS3.CR30NtDFR2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8772804/v1/2b7d18f1bd6b6f769c731039.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Coupling FT-Mediated Speed Breeding and CRISPR/Cas9 for Rapid Trait Improvement in Nicotiana tabacum","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe acceleration of crop improvement cycles is a fundamental objective of modern agriculture, driven by the urgent need to adapt global food systems to a rapidly changing climate and expanding population. While recent advances in genomic technologies have provided the tools to identify and engineer beneficial alleles, the rate of genetic gain is ultimately limited by the biological generation time of the target species (Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This limitation is particularly acute in perennial crops and woody species, where the juvenile can span from several years to over a decade (Flachowsky et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Prado et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Consequently, the introgression of a single edited trait into a commercial perennial cultivar via conventional backcrossing can be prohibitively slow and resource-intensive.\u003c/p\u003e \u003cp\u003eAlthough genome editing technologies, particularly CRISPR/Cas systems, have revolutionized trait development (Tuncel et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Tuncel et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) their application in perennials remains constrained by the time required to generate mature, edited plants.To address this, \"speed breeding\" methodologies manipulating photoperiod and temperature were developed (Watson et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, environmental speed breeding is often ineffective for species with complex dormancy requirements or obligate short-day responses. For such recalcitrant species, genetic intervention using the \u003cem\u003eFLOWERING LOCUS T\u003c/em\u003e (\u003cem\u003eFT\u003c/em\u003e) gene offers a potent alternative. \u003cem\u003eFT\u003c/em\u003e encodes florigen, a conserved mobile protein that initiates floral development (Corbesier and Coupland \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Wickland and Hanzawa \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Overexpression of \u003cem\u003eFT\u003c/em\u003e induces precocious flowering in diverse genera, including \u003cem\u003ePopulus\u003c/em\u003e, \u003cem\u003eMalus\u003c/em\u003e, and \u003cem\u003eCitrus Citrus\u003c/em\u003e (B\u0026ouml;hlenius et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Klocko et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Adeyemo et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Khan et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eDespite its utility, constitutive \u003cem\u003eFT\u003c/em\u003e overexpression in a final crop product is undesirable due to potential pleiotropic effects on yield and architecture. Furthermore, the persistence of the transgene triggers stringent regulatory oversight (Strauss et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Buchholzer and Frommer \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, a unified system that utilizes \u003cem\u003eFT\u003c/em\u003e to accelerate the breeding cycle during editing, but allows for subsequent transgene removal, would combine the speed of genetic induction with the regulatory advantages of transgene-free editing. Here, we establish such a system in \u003cem\u003eNicotiana tabacum\u003c/em\u003e, utilizing an integrated \u003cem\u003eAtFT\u003c/em\u003e overexpression and CRISPR/Cas9 module to achieve rapid generation cycling and efficient editing of the \u003cem\u003eNtDFR\u003c/em\u003e gene family.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant Materials and Growth Conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (ecotype Col-0) and tobacco (\u003cem\u003eNicotiana tabacum\u003c/em\u003e cv. K326) were used in this study. All plants were cultivated in a controlled environment growth chamber maintained under a 16:8 h light/dark photoperiod at 24\u0026ndash;28\u0026deg;C.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eVector constructions\u003c/h3\u003e\n\u003cp\u003eThe full-length coding sequence (CDS) of \u003cem\u003eAtFT (AT1G65480)\u003c/em\u003e was amplified from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e floral bud tissues using specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and Q5 high-fidelity DNA polymerase (M0491L, NEB). The 528 bp amplicon was cloned into the pMDC43 binary vector (Curtis and Grossniklaus \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) between the \u003cem\u003eKpn\u003c/em\u003eI and \u003cem\u003eSac\u003c/em\u003eI restriction sites, placing it under the CaMV 35S promoter to generate pMDC-AtFT. The plasmid was confirmed by Sanger sequencing.\u003c/p\u003e \u003cp\u003eTo create the integrated editing vector, the \u003cem\u003eCaMV35S::AtFT::Nos\u003c/em\u003e cassette was amplified from pMDC-AtFT and subcloned into the pFGC-Cas9-GW backbone (Nguyen et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) at the \u003cem\u003eSpe\u003c/em\u003eI site via Gibson Assembly NEBuilder\u0026reg; HiFi DNA Assembly Cloning Kit (E5520S, NEB ) (Gibson et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The plasmid was confirmed by Sanger sequencing. The entry vector, pAtU6-GW, driven by the \u003cem\u003eAtU6\u003c/em\u003e promoter and flanked by \u003cem\u003eattL1-attL2\u003c/em\u003e sequence was synthesized by Twist Bioscience (CA, USA).\u003c/p\u003e \u003cp\u003eFor gRNA design, the web tool, CCTop (Stemmer et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) was used to design. A 23 nucleotides sequence that is identical in \u003cem\u003eNtDFR1\u003c/em\u003e (Genebank: EF421429) and \u003cem\u003eNtDRF2\u003c/em\u003e (Genebank: EF421430) genes at the first exon of both two genes was selected. DNA oligonucleotide pairs (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) was synthesized by PhusaGenomics (Can Tho, Vietnam) and annealed to generate dimer. The dimer was cloned into \u003cem\u003eBbsI\u003c/em\u003e sites of pAtU6-GW to create pAtU6-NtDFR, which was then mobilized into the binary vector, pFGC-Cas9-AtFT-GW, at the \u003cem\u003eattR1-attR2\u003c/em\u003e site using Gateway\u0026trade; LR Clonase (Invitrogen) to create pFGC-Cas9-AtFT-NtDRF.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAgrobacterium\u003c/b\u003e \u003cb\u003e-mediated transformation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe binary vectors were electroporated into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101. Tobacco leaf disk transformation was performed as described by (Horsch et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1985\u003c/span\u003e) with modifications. Leaf explants were infected with bacterial suspension (OD\u003csub\u003e600\u003c/sub\u003e=0.5) for 30 minutes and co-cultivated for 48 hours in the dark. Explants were subsequently selected on Murashige and Skoog (MS) medium supplemented with 500 mg/L cefotaxime and either 20 mg/L hygromycin (for pMDC-AtFT) or 3 mg/L glufosinate (for pFGC-Cas9-AtFT-NtDFR).\u003c/p\u003e\n\u003ch3\u003eMolecular and Phenotypic Characterization\u003c/h3\u003e\n\u003cp\u003eChromosomal DNA was isolated following routine isolation techniques from young leaf tissues of the overexpression or CRISPR/Cas9 transgenic lines. The PCR amplification was performed using gene-specific primers with Q5 High fidelity DNA polymerase (M0491L, NEB). Primers used for CRISPR genotyping were listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. PCR products were extracted and purified from the agarose gel using GeneJET Gel Extraction Kit (K0691, Thermo Scientific), subsequently cloned into pJET1.2 Cloning Vector (K1231, Thermo Scientific). Individual clones were sequenced by Sanger sequencing using the pJET1.2-F and pJET1.2-R primers. For CAPS assay, the PCR products were incubated with \u003cem\u003ePvuII\u003c/em\u003e enzyme (R0151S, NEB) at 37 \u003csup\u003e0\u003c/sup\u003eC overnight. After digestion, the mixtures were loaded in 2.5% agarose gel.\u003c/p\u003e \u003cp\u003eShoot apices from control and overexpression lines at 15 days after germination were collected. RNA was extracted using TRIzol reagent (Invitrogen), purified with FastPure Plant Total RNA Isolation Kit (Vazyme, China) and treated with TURBO\u0026trade; DNase following the manufacturer's instructions. cDNA was synthesized using oligo(dT) primers (15-mer) and HiScript IV 1st Strand cDNA Synthesis Kit (Vazyme, China) following the manufacturer's instructions. Transcript levels of genes were determined by qRT-PCR using an QuantStudio 5 real-time PCR following the SYBR Green method (Applied Biosystems). Gene expression levels were normalized to the expression of the Actin housekeeping gene and analyzed with 2\u003csup\u003e\u0026minus;ΔΔ\u003cem\u003eC\u003c/em\u003eT\u003c/sup\u003e method (Livak and Schmittgen \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Primers used for qRT-PCR were listed in Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eTotal anthocyanin content was determined according to the method described by Weiss and Halevy (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Briefly, corollas were snap-frozen in liquid nitrogen and stored at -80\u0026deg;C until analysis. A total of 100 mg of powdered samples was incubated in 600 \u0026micro;L of extraction buffer (methanol containing 1% HCl) in the dark for 24 hours at 4\u0026deg;C. The addition of 200 \u0026micro;L of water and 200 \u0026micro;L of chloroform was followed by centrifugation at 12,000 \u0026times; g for 10 minutes at 4\u0026deg;C. Absorbance of the supernatant was measured at 530 nm (A530) and 657 nm (A657) using microplate reader. Total anthocyanin content was calculated based on the molar absorbance of cyanidin-3-O-glucoside, with A530\u0026ndash;0.25 \u0026times; A657 used for quantification. Each sample was extracted and measured in five biological independent experiments.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eSample means between genotypes were compared using Student's \u003cem\u003et\u003c/em\u003e-test or one-way ANOVA followed by Tukey's HSD test. All statistical analyses were done using GraphPad Prism 9.0 (GraphPad Software, Inc.)\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussions","content":"\u003cp\u003e \u003cb\u003eEctopic Expression of\u003c/b\u003e \u003cb\u003eAtFT\u003c/b\u003e \u003cb\u003eAccelerates Floral Transition in Tobacco\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo examine the effect of ectopic expression FT on growth and development of the plant, we analyzed tobacco lines transformed with the \u003cem\u003eAtFT\u003c/em\u003e overexpression vector (pMDC-AtFT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Five independent transgenic events were generated and confirmed via PCR genotyping (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Important, the T0 transgenic lines produced flowers under \u003cem\u003ein vitro\u003c/em\u003e conditions (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). To evaluate the impact of FT on plant and growth of the transgenic lines, T1 plants from two independent overexpression lines (OX6 and OX8) were sown and observed. While wild-type (WT) K326 plants required approximately 12 weeks to initiate flowering under long-day conditions, \u003cem\u003eAtFT\u003c/em\u003e-overexpressing lines (OX6 and OX8) produced visible floral buds as early as 3 weeks post-germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), and set fruit within 3 weeks after germination (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). This represents a four-fold acceleration of the generation cycle. Importantly, this rapid development did not compromise reproductive viability. The transgenic lines produced viable seeds (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC), a prerequisite for any breeding application. The plants exhibited a short stature solely due to the extreme reduction in the vegetative growth phase; however, germination rates and seedling vigor remained comparable to the wild type (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo mechanistically validate that the observed phenotype was driven by the integration of the heterologous AtFT into the tobacco floral network, we quantified the expression of endogenous floral identity genes. \u003cem\u003eNtSOC1\u003c/em\u003e (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1) and \u003cem\u003eNtAP1\u003c/em\u003e (APETALA1) are key integrators acting downstream of the florigen signal (Lee and Lee \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Our qRT-PCR analysis revealed a significant upregulation of both \u003cem\u003eNtSOC1\u003c/em\u003e and \u003cem\u003eNtAP1\u003c/em\u003e in the shoot apices of transgenic plants at 12 days post-germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). This molecular evidence confirms that \u003cem\u003eAtFT\u003c/em\u003e acts as a potent floral activator in \u003cem\u003eN. tabacum\u003c/em\u003e, effectively bypassing the juvenile phase requirements.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSimultaneous Editing and speed breeding using the\u003c/b\u003e \u003cb\u003eAtFT\u003c/b\u003e\u003cb\u003e-CRISPR System\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHaving validated the speed breeding component, we deployed the integrated vector pFGC-Cas9-AtFT-NtDFR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) to test the system's capacity for simultaneous rapid cycling and genome editing. The \u003cem\u003eNtDFR\u003c/em\u003e genes were selected as a target because disruption of anthocyanin biosynthesis yields a readily scorable white-flower phenotype. To simultaneously mutate both \u003cem\u003eDFR\u003c/em\u003e genes in tobacco, a guide RNA targeting the first exon of both genes was selected to construct the pFGC-Cas9-\u003cem\u003eAtFT\u003c/em\u003e-\u003cem\u003eNtDFR\u003c/em\u003e binary vector. Importantly, the single guide RNA (sgRNA) was designed to overlap a \u003cem\u003ePvu\u003c/em\u003eII restriction site, facilitating rapid genotypic screening of edited lines via Cleaved Amplified Polymorphic Sequences (CAPS) analysis. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation of the tobacco cultivar K326 generated four basta-resistant lines (CR7, CR30, CR31, and CR39) that carried \u003cem\u003eAtFT\u003c/em\u003e and CRISPR/Cas9 components (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). To examine the editing efficacy of Cas9 in the speed breeding complex, these four transgenic lines were subjected to genotyping using the PCR-CAPS assay with \u003cem\u003ePvu\u003c/em\u003eII. While the wild-type amplicon was completely digested by the \u003cem\u003ePvu\u003c/em\u003eII restriction enzyme, all four transgenic events showed resistance to \u003cem\u003ePvu\u003c/em\u003eII digestion in either \u003cem\u003eNtDFR1\u003c/em\u003e, \u003cem\u003eNtDFR2\u003c/em\u003e, or both (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The CAPS assay analysis suggested that the transgenic lines CR7 and CR39 were heterozygous for mutations in both \u003cem\u003eNtDFR1\u003c/em\u003e and \u003cem\u003eNtDFR2\u003c/em\u003e genes, whereas the edited lines CR30 and CR31 possessed heterozygous mutations only in the \u003cem\u003eNtDFR1\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Sanger sequencing was used to further characterize the mutants CR7 and CR30, confirming that mutant line CR7 possessed a 3-bp and 3-bp deletion for \u003cem\u003eDFR1\u003c/em\u003e and \u003cem\u003eDFR2\u003c/em\u003e respectively, while a 2-bp deletion was obtained in the edited line CR30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These results suggest that the gene editing efficacy of the Cas9 system in this accelerated cycle complex is as high as in the original binary backbone without \u003cem\u003eAtFT\u003c/em\u003e (Do et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Nguyen et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next examined the inheritance of the mutations in the T1 generation. Plants from the two edited lines, CR7 and CR30, were subjected to CAPS assays for genotyping. Gel electrophoresis results confirmed that the mutations identified in the T0 generation were successfully transmitted to the next generation (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Consistent with the genetic characteristics of the T0 progenitors (CR7: \u003cem\u003eDFR1/dfr1\u003c/em\u003e, \u003cem\u003eDFR2/dfr2\u003c/em\u003e; CR30: \u003cem\u003eDFR1/dfr1\u003c/em\u003e, \u003cem\u003eDFR2/DFR2\u003c/em\u003e), T1 progeny from the CR7 line segregated to produce single mutants for \u003cem\u003edfr1\u003c/em\u003e and \u003cem\u003edfr2\u003c/em\u003e, while only single \u003cem\u003edfr1\u003c/em\u003e mutants were obtained from CR30. Notably, we were able to successfully recover homozygous double mutants (\u003cem\u003edfr1 dfr2\u003c/em\u003e) in the T1 generation derived from CR7. As \u003cem\u003eNtDFR\u003c/em\u003e is known to determine flower color in tobacco (Kazama et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Lim et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), we analyzed floral pigmentation and quantified total anthocyanin content in the \u003cem\u003edfr\u003c/em\u003e mutant lines. While all plants carrying \u003cem\u003eAtFT\u003c/em\u003e flowered early (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), variations in flower color were strictly correlated with the presence of specific mutations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). While \u003cem\u003edfr1\u003c/em\u003e single mutants produced pale pink flowers, \u003cem\u003edfr2\u003c/em\u003e single mutants produced white flowers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA). Anthocyanin levels extracted from corolla tissues of fully expanded flowers showed consistency with visible petal color intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB). The anthocyanin content of \u003cem\u003edfr1\u003c/em\u003e single mutants was reduced by approximately half compared to wild-type plants, while \u003cem\u003edfr2\u003c/em\u003e single mutants contained 90% and 72% less anthocyanin than WT and \u003cem\u003edfr1\u003c/em\u003e single mutants, respectively. These results confirm that \u003cem\u003eNtDFR1\u003c/em\u003e and \u003cem\u003eNtDFR2\u003c/em\u003e are responsible for the color phenotype in tobacco flowers and suggest that \u003cem\u003eNtDFR2\u003c/em\u003e is the major contributor to the pink color, as has been reported in tobacco cv. Xanthi (Kazama et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn summary, our study establishes a robust integrated speed-breeding system that couples the floral induction of \u003cem\u003eAtFT\u003c/em\u003e with the precision of CRISPR/Cas9 editing. We demonstrated that this dual-function system can compress the breeding cycle of \u003cem\u003eN. tabacum\u003c/em\u003e from months to weeks while achieving high-efficiency mutagenesis of the \u003cem\u003eNtDFR\u003c/em\u003e loci. Crucially, the independent segregation of the editing machinery and the targeted mutation in the T1 generation allows for the rapid recovery of transgene-free, homozygous edited lines. This capability is particularly significant for overcoming the regulatory and biological barriers associated with the genetic improvement of woody perennials and crops with extended juvenile phases, providing a versatile toolkit for modern crop breeding.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by the Vietnam Academy of Science and Technology, and the Russian Science Foundation in a bilateral project coded QTRU06.08/24\u0026ndash;26 (or 24-44-04007).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eCXN and PTD conceived and conceptualized the study. CXN designed, performed the experiments, and analyzed data. AMN performed experiments. PTD managed the project. CXN wrote the first draft of the manuscript, which was subsequently revised and proofread by all authors.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank PlantgenSolutions for providing tobacco transformation services.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll materials reported in this paper will be shared by the corresponding authors upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdeyemo OS, Chavarriaga P, Tohme J, Fregene M, Davis SJ, Setter TL (2017) Overexpression of Arabidopsis FLOWERING LOCUS T (FT) gene improves floral development in cassava (Manihot esculenta, Crantz). 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Mol Plant 8:983\u0026ndash;997. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.molp.2015.01.007\u003c/span\u003e\u003cspan address=\"10.1016/j.molp.2015.01.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"functional-and-integrative-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fige","sideBox":"Learn more about [Functional \u0026 Integrative Genomics](http://link.springer.com/journal/10142)","snPcode":"10142","submissionUrl":"https://submission.nature.com/new-submission/10142/3","title":"Functional \u0026 Integrative Genomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Speed breeding, CRISPR/Cas9, FLOWERING LOCUS T, flower color, DFR","lastPublishedDoi":"10.21203/rs.3.rs-8772804/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8772804/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe commercial deployment of genome-edited crops is frequently bottlenecked by the extended juvenile phases of perennial species and the complex regulatory landscapes governing plants with integrated exogenous DNA. Speed breeding protocols, which utilize environmental manipulation to accelerate development, have proven effective for annual cereals but often lack efficacy in perennials. In this study, we report a genetic speed breeding system that couples the overexpression of the floral integrator \u003cem\u003eFLOWERING LOCUS T\u003c/em\u003e (\u003cem\u003eFT\u003c/em\u003e) with CRISPR/Cas9-mediated genome editing. Using \u003cem\u003eNicotiana tabacum\u003c/em\u003e as a model for polyploid crops, we demonstrate that constitutive expression of \u003cem\u003eArabidopsis thaliana FT\u003c/em\u003e (\u003cem\u003eAtFT\u003c/em\u003e) reduces the generation time from 12 weeks to approximately 3 weeks without compromising fertility or seed viability. To leverage this acceleration for trait improvement, we engineered a binary vector co-expressing \u003cem\u003eAtFT\u003c/em\u003e and a CRISPR/Cas9 cassette targeting the \u003cem\u003eNtDFR\u003c/em\u003e (\u003cem\u003edihydroflavonol 4-reductase\u003c/em\u003e) loci. This integrated system induced rapid flowering and simultaneous disruption of anthocyanin biosynthesis, yielding \u003cem\u003entdfr\u003c/em\u003e mutants with a distinct white-flower phenotype. Importantly, the edited alleles were heritable, while the \u003cem\u003eFT-Cas9\u003c/em\u003e transgene could be segregated out in the next generation, restoring the wild-type photoperiodic response in the edited progeny. This transgenic facilitator approach offers a scalable platform for the rapid introgression of edited traits into recalcitrant crop species, potentially reducing the breeding cycle of perennials from years to months.\u003c/p\u003e","manuscriptTitle":"Coupling FT-Mediated Speed Breeding and CRISPR/Cas9 for Rapid Trait Improvement in Nicotiana tabacum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-02 03:17:25","doi":"10.21203/rs.3.rs-8772804/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-04T23:45:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-08T02:00:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-02T02:27:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"65197687987206054349670546011482870585","date":"2026-03-24T02:16:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"207830772875183437191045830653005281403","date":"2026-03-23T01:36:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-22T00:26:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-09T08:36:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-07T07:24:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Functional \u0026 Integrative Genomics","date":"2026-02-03T07:38:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"functional-and-integrative-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fige","sideBox":"Learn more about [Functional \u0026 Integrative Genomics](http://link.springer.com/journal/10142)","snPcode":"10142","submissionUrl":"https://submission.nature.com/new-submission/10142/3","title":"Functional \u0026 Integrative Genomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f6e07841-0165-4641-ad02-13ea4d6f200e","owner":[],"postedDate":"March 2nd, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-04T23:45:24+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T23:53:50+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-02 03:17:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8772804","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8772804","identity":"rs-8772804","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}