Establishing a Tissue Culture–based Transformation System for Cheatgrass (Bromus tectorum L.) to Enable Functional Genomic Studies

Establishing a Tissue Culture–based Transformation System for Cheatgrass (Bromus tectorum L.) to Enable Functional Genomic Studies | 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 Establishing a Tissue Culture–based Transformation System for Cheatgrass (Bromus tectorum L.) to Enable Functional Genomic Studies Shahbaz Ahmed, Jonah Mensonides, Ian Burke This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8137674/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Cheatgrass ( Bromus tectorum L.) is one of the most invasive annual grasses in the western United States. It has spread extensively across rangelands and cropping systems and increasingly expresses herbicide resistance. Despite its ecological and agricultural significance as a major invasive weed, functional genetic studies in cheatgrass have been hindered by the absence of a reproducible transformation system. Here, we report the first successful protocol for Agrobacterium tumefaciens –mediated genetic transformation of cheatgrass. Mature and immature embryos were evaluated as explant sources to optimize callus induction and regeneration. Callus formation from immature embryos began earlier than from mature embryos, which required a longer culture period. Hygromycin-resistant calli expressing green fluorescent protein (GFP) were obtained from both explant types, confirming successful genetic transformation. Following Agrobacterium transformation, calli derived from immature embryos regenerated green, viable shoots, whereas those from mature embryos frequently produced bleached, chlorophyll-deficient tissues. PCR amplification of GFP and hygromycin phosphotransferase (HPT) confirmed stable integration of transgenes into the cheatgrass genome. Our work presents the first reproducible immature embryo–based transformation system for cheatgrass, enabling molecular genetics and functional genomics studies in weedy grasses. Bromus tectorum cheatgrass Downy brome Agrobacterium tumefaciens tissue culture GFP herbicide resistance molecular weed biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key Message The study establishes the first transformation system for cheatgrass, a highly adaptive and invasive grass, enabling molecular research on herbicide resistance and environmental adaptation. Introduction Weeds represent one of the greatest challenges to global agriculture, reducing crop yields and increasing production costs across diverse cropping systems (Horvath et al., 2023 ). Among them, cheatgrass or downy brome ( Bromus tectorum L.) is a highly invasive annual grass that has spread extensively across North America and other regions (Bradley et al., 2018 ). It dominates rangelands, wheat-based systems, and disturbed habitats, where its success is driven by prolific seed production, seed dormancy, rapid establishment, and tolerance to multiple stresses (Molvar et al., 2024). In addition, the evolution of herbicide resistance, including resistance to glyphosate, further complicates its management (Geddes & Pittman, 2022 ; Revolinski et al., 2023 ; Ribeiro, 2025 ). Despite its ecological and agronomic importance, molecular and genetic studies on cheatgrass remain limited, largely due to the absence of reproducible transformation and regeneration protocols. Plant transformation technologies have revolutionized crop research by enabling functional gene studies, trait engineering, and genome editing (P. Wang et al., 2025 ). While well established for model plants such as Arabidopsis thaliana and major crops such as rice, maize, and wheat (Clough & Bent, 1998 ; Hayta et al., 2019 ; Ishida et al., 2007 ; Sahoo et al., 2011 ), these approaches have rarely been extended to weedy species. Bringing weeds into the molecular biology laboratory has the potential to transform our understanding of weed biology, herbicide resistance, and adaptation (Wong et al., 2022 ). Developing robust tissue culture and transformation protocols for weedy species would open new avenues for dissecting the genetic mechanisms underlying invasiveness and resistance, and for testing biotechnological strategies for weed management (Mellado-Sánchez et al., 2020a ). Tissue culture–based transformation, particularly through Agrobacterium tumefaciens –mediated delivery of transgenes, provides a practical and widely used system for stable genetic modification (Gelvin, 2003 ). However, transformation efficiency depends strongly on explant type, callus induction, regeneration capacity, and selection regime (Dhar & Joshi, 2005 ; Ziemienowicz, 2014 ). In grasses, particularly non-model species, recalcitrance to transformation has been a persistent challenge. One contributing factor is that Agrobacterium -mediated gene delivery elicits host immune responses, which can inhibit T-DNA transfer and integration (Luo et al., 2025 ). Non-domesticated weed species often exhibit heightened stress resilience and stronger innate defense responses compared with domesticated crops, further reducing their amenability to transformation (Luo et al., 2025 ; Mellado-Sánchez et al., 2020a ). To date, no transformation system has been reported for cheatgrass. In this study, we developed the first Agrobacterium -mediated transformation protocol for cheatgrass. We optimized callus induction from both mature and immature embryos, evaluated their suitability for transformation, and demonstrated stable green fluorescent protein (GFP) expression in calli and regenerated plants. Molecular confirmation by PCR further validated integration of the transgenes. The work establishes a foundation for molecular genetic studies in cheatgrass and represents an important step toward bringing weedy species into the molecular biology laboratory. Materials and Methods Plant Material and Seed Preparation Seeds of cheatgrass were obtained from a winter-type population maintained under greenhouse conditions, originally collected from Pomeroy, WA. Two types of seed were used: mature seeds and immature seeds. Mature seeds were previously harvested and stored at room temperature in the laboratory. For immature seeds, florets were harvested at early developmental stages before the spikelet turned purple. Seeds were surface sterilized in 10% sodium hypochlorite for 5 min, followed by three rinses with sterile distilled water. Embryo Isolation and Callus Induction Sterilized mature seeds were pre-soaked on moistened sterile filter paper for 30 min to facilitate embryo dissection. Embryos were excised under a stereomicroscope in a laminar flow hood and placed scutellum-side down on callus induction medium (CIM). CIM consisted of 4.43 g/L Murashige and Skoog (MS) salts (M519), 30 g/L maltose (M588), and 3.5 g/L Gelzan™ (G3251) (all from PhytoTechnology Laboratories, Lenexa, KS, USA). The medium was adjusted to pH 5.8, autoclaved, and allowed to cool to approximately 50°C before adding filter-sterilized supplements: 2.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D; D309), 1.25 mg/L CuSO₄ (C375), 1 g/L casein hydrolysate (C184), and 10 mL/L CI vitamin stock (100 mg/L thiamine HCl (T390), 35 g/L myo-inositol (I703), and 69 g/L L-proline (P698), prepared according to (Hinchliffe & Harwood, 2019a ). All components were obtained from PhytoTechnology Laboratories, and the vitamin stock was prepared in advance, filter-sterilized, and stored at 4°C. Immature embryos were isolated from florets under the microscope at the late milky to early dough stage, when embryos were firm and translucent but before seed coat pigmentation began. Immature embryos were excised using forceps and a teasing needle, then placed scutellum-side down on CIM. Plates were incubated in the dark at 26–28°C for at least 6–8 weeks. Actively dividing calli were subcultured every two weeks by fragmentation onto fresh CIM to maintain proliferation. Plasmid Construction and Agrobacterium Transformation The empty binary vector pGFPGUSPlus ((Plasmid #64401, addgene, Watertown, MA USA), carrying hygromycin resistance, GFP, and GUS reporter genes under the CaMV 35S promoter, was introduced into Agrobacterium tumefaciens strain GV3101 (rifampicin- and gentamycin-resistant) using a heat-shock method. Competent cells were incubated with 3 µL plasmid DNA on ice for 30 minutes, flash-frozen in liquid nitrogen for 1 min, thawed at 37°C for 2 min, and recovered in 1 mL YEP broth at 28°C for 3 h. Aliquots (50–150 µL) were plated on YEP agar containing tryptone 10g/L, yeast extract 10g/L, sodium chloride 5g/L, pH7.2, agar 15g/L, 50 mg/L gentamycin and 50 mg/L kanamycin and incubated at 28°C for 48 h. Preparation of Agrobacterium Culture for Infection A single transformed colony was inoculated into 3 mL YEP broth (YEP without agar) with 50 mg/L kanamycin and 50 mg/L gentamycin antibiotics and grown overnight at 28°C with shaking. Glycerol stocks were prepared (700 µL culture + 300 µL 50% glycerol) and stored at − 80°C. For infection, Cells harboring the pGFPGUSPlus plasmid from glycerol stocks were streaked onto MG/L plates (10 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, 15 g/L agar; pH 7.2) with antibiotics and incubated at 28°C for 24–48 h. Cells were harvested, resuspended in CIM to an OD₆₀₀ of 0.6, and supplemented with 200 µM acetosyringone. Agrobacterium -Mediated Callus Transformation Actively proliferating calli were immersed in the bacterial suspension for 10 min with occasional shaking, blotted on sterile filter paper, and co-cultivated in the dark at 28°C for 3 days. Calli were then transferred to CIM supplemented with 150 mg/L timentin (for Agrobacterium elimination) and 40 mg/L hygromycin (for selection) and incubated at 26–28°C in dark for 2 weeks. After two weeks, the calli was transferred to fresh media for two more weeks with 30 mg/L hygromycin and 150 mg/L timentin. Shoot Regeneration and Rooting Hygromycin-resistant calli were transferred to regeneration medium (4.43 g/L MS salts, 30 g/L maltose, 3.5 g/L Gelzan, 1 mg/L BAP, 0.5 mg/L kinetin, 10 mL/L T/R vitamin stock (Hinchliffe & Harwood, 2019a ), 150 mg/L timentin, and 20 mg/L hygromycin) and incubated at 25°C under a 16 h light/8 h dark photoperiod. Regenerated shoots were transferred to rooting medium for 2–3 weeks (½-strength MS salts, 30 g/L maltose, 2g/L Gelzan, 0.5 mg/L indole-3-butyric acid, 15 mg/L hygromycin). Rooted plants were acclimatized in Sunshine Mix 4 (LA4 aggregate) soil in the greenhouse. GFP Fluorescence Screening GFP signals in calli and regenerated plants were detected using a Leica MDG41 fluorescence stereomicroscope. Samples were first observed under brightfield illumination to assess tissue morphology and then re-examined using a GFP filter set (excitation 450–490 nm; emission 500–550 nm). To minimize the possibility of misinterpreting autofluorescence, imaging was performed under complete darkness, and tissues were examined sequentially under multiple filters sets such as white light, UV and mCherry to determine if the signal is present under all filters or just GFP. For regenerated plants, detached leaves were placed on glass slides and visualized under the same filter conditions. GFP-positive tissues were identified by distinct green fluorescence, which was documented using the integrated Leica digital camera system. Genomic DNA Extraction and PCR Confirmation Leaf tissue (1–2 cm) from GFP-positive putative T0 at four leaves stages growing in soil was collected, and genomic DNA was extracted using the MagMax DNA Extraction Kit (Thermo Fisher Scientific) following the manufacturer’s instructions on the KingFisher Apex (Thermo Fisher Scientific) automated system. PCR amplification was performed using GreenDream Taq PCR Master Mix (Thermo Fisher Scientific) with primer pairs designed to amplify two transgene regions: a 244 bp fragment spanning the CaMV 35S promoter and GFP junction, and a 226 bp fragment spanning the CaMV 35S promoter and the hygromycin phosphotransferase (HPT) gene. Wild-type genomic DNA and plasmid DNA were included as negative and positive controls, respectively, alongside a no-template control. Amplification was performed on a Bio-Rad T100 thermal cycler under the following program: initial denaturation at 95°C for 3 min; 34 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min. PCR products were resolved on 2% agarose gels stained with SYBR Safe DNA stain (Thermo Fisher Scientific) and visualized under UV illumination. Results Callus formation from Mature and Immature Embryos Both mature and immature embryos were evaluated as explant sources. In mature embryos, callus formation occurred in two distinct phases. During the initial phase, a soft, non-proliferative tissue developed within two weeks, with no further growth observed over the following 4–6 weeks. Subsequently, actively dividing calli were initiated, which required an additional 2–3 weeks to attain sufficient size and vigor. Upon subculturing, these proliferating calli exhibited a 100% regrowth rate. Overall, approximately 15–20% of mature embryos successfully generated actively growing calli (Fig. 1 A–C). Callus formation from immature embryos was faster, with actively proliferating calli observed from the beginning of the culture period, and full-sized calli obtained within 6–8 weeks. However, the efficiency of healthy callus induction was lower, with less than 10% of immature embryos producing viable calli (Fig. 2 A). Once established, these calli maintained 100% growth upon subculture and provided abundant material for subsequent regeneration and transformation experiments (Fig. 1 D-E). Agrobacterium -mediated Transformation Both mature- and immature-derived calli were amenable to Agrobacterium tumefaciens -mediated transformation using a binary vector carrying GFP and a hygromycin resistance marker. Stable GFP fluorescence was detected in sectors of hygromycin-resistant calli derived from both types of embryos and persisted after subculturing (Fig. 2 A-F). Regeneration of Transgenic Plants To establish baseline regeneration efficiency, non-transformed calli derived from both mature and immature embryos were first evaluated. Under regeneration conditions, up to 50% of healthy, proliferating calli from mature and immature embryo regenerated into green, growing shoots (Fig. 3 A). These shoots readily produced roots in rooting medium and acclimatized successfully to soil in the greenhouse (Fig. 3 B). However, calli derived from mature embryos exhibited a sharp decline in regeneration efficiency on selection media following Agrobacterium transformation, yielding primarily bleached, chlorophyll-deficient shoots under hygromycin selection (Fig. 3 C) In comparison, immature embryo-derived calli post Agrobacterium transformation showed superior regeneration potential. Under selection, small greening spots appeared on immature embryo derived calli, which gradually developed into shoots that elongated and remained healthy on hygromycin-containing medium. These shoots subsequently formed strong root systems on rooting medium and acclimatized successfully when transferred to soil (Fig. 3 D-G). Molecular Confirmation of Transgene Integration Regenerated shoots exhibited distinct localized GFP fluorescence signals that were absent in non-transgenic controls, and fluorescence remained detectable in the leaves of fully regenerated plants, confirming transgene expression in mature tissues (Fig. 4 A–H). Amplification through PCR of junction fragments between the CaMV 35S promoter and either the GFP or hygromycin phosphotransferase (HPT) gene yielded the expected amplicons in all putative transgenic lines. No products were detected in wild-type or no-template controls, whereas plasmid DNA produced the expected fragments (Fig. 5 ). Discussion The study presents the first successful protocol for Agrobacterium -mediated genetic transformation of cheatgrass, a highly invasive and agriculturally significant weed. By combining embryo-derived callus induction, optimized selection regimes, and regeneration assays, we demonstrate that cheatgrass is amenable to tissue culture–based transformation, thereby establishing a platform for molecular genetic studies in this species. Callus induction, and agrobacterium transformation We detect clear differences in callus induction and regeneration between mature and immature embryos. In mature embryos, the initial soft tissue stage represents limited dedifferentiation before a subset of responsive cells re-enters the cell cycle and generates actively dividing calli. Similar to the results reported by (Nakasha et al., 2016 ), increasing 2,4-D concentration in our study did not enhance callus formation in mature embryos (data not shown). This is likely because mature embryos are composed of differentiated, less mitotically active cells, which are slower to respond to auxin-rich induction media (Ganeshan et al., 2003 ; Suo et al., 2021 ). Once established, however, subculture of actively growing calli from mature embryos consistently generates sufficient material for downstream applications, including transformation and shoot regeneration. Immature embryos, excised from florets before visible purple pigmentation, initiate actively dividing calli from the start but at lower frequency. This reduction is due to several factors. First, florets at very early stages are watery, making excision technically difficult and increasing the likelihood of mechanical damage. Second, embryo size is small compared with other crops such as maize, wheat, or Brachypodium , and slight variation in developmental stage introduces heterogeneity in response. Similar trade-offs between embryo developmental stage, callus induction frequency, and regeneration potential have been reported in cereals such as barley and wheat (Hinchliffe & Harwood, 2019b ; Ishida et al., 2015 ). Despite the lower initial frequency, once actively growing calli is established from immature embryos, subculture produces fresh, proliferating callus at nearly 100% efficiency. Continuous subculturing provides ample material for transformation experiments. Once actively growing calli was obtained, both mature-derived and immature-derived tissues were amenable to Agrobacterium transformation. Putative transgenic calli maintained active growth and were readily identified under GFP filters. Autofluorescence is common in grass tissues due to chlorophyll and phenolic compounds, and most calli display a dull green background under blue light (Billinton & Knight, 2001 ; Donaldson, 2020 ). However, true GFP-positive calli emit strong, localized signals that are readily distinguished from background when samples are observed first under brightfield illumination, mcherry and then under GFP filters in complete darkness (Mellado-Sánchez et al., 2020b ). The persistence of GFP signals through multiple rounds of subculture provides strong evidence for stable T-DNA integration. Selection and regeneration bottlenecks A major bottleneck of this system is the reduced transformation efficiency and regeneration capacity of transgenic calli. Non-transformed calli regenerate green shoots at relatively high frequency, whereas only a small fraction of hygromycin-resistant calli from immature embryos produce viable shoots due to selection indicating lower transformation efficiency. The lower transformation efficiency in non-domesticated plant species is due to their increased immunity against biotic and abiotic stresses (Luo et al., 2025 ; Singh & van der Knaap, 2022 ). Calli derived from mature embryos regenerated even less efficiently post agrobacterium transformation (Ward & Jordan, 2001 ), often producing bleached, chlorophyll-deficient shoots. In our system, the duration of culture may be particularly critical: mature embryos require 6–8 weeks to produce actively dividing calli, followed by additional rounds of subculture to generate sufficient material, before transformation and selection steps are imposed. As a result, regenerated shoots from mature embryo calli are derived from tissues that have already spent 10–12 weeks on callus induction medium, potentially reducing regenerative capacity. By the time transformed calli are transferred to regeneration medium, the tissue may be too old to support the development of green shoots (Chakravarty & Goswami, 1999 ; Orshinsky & Tomes, 1985 ). In immature embryo-derived calli, the shorter culture duration before transformation likely contributes to more successful regeneration into green shoots. However, we also observe that shoots emerging from immature calli must remain in direct contact with regeneration medium; those that grow above the surface eventually senesce (Mazumdar et al., 2010 ). This problem can be alleviated by frequent transfer to fresh medium, ensuring that developing shoots maintain contact. Root formation occurs more efficiently when regenerated shoots are transferred to a reduced selective rooting medium, a strategy consistent with protocols in barley and rice where selective pressure is relaxed during late developmental stages (Hayta et al., 2019 ). Transplanted plantlets establish well in soil, producing fertile T0 plants. Conclusion The ability to stably transform cheatgrass has significant implications for weed science. Weeds remain largely excluded from the molecular biology toolkit, despite their major role in agricultural yield loss and herbicide resistance evolution. As emphasized in the biotechnological roadmap for weed management (Wong et al., 2022 ), transformation and functional genomics are prerequisite tools for dissecting the genetic basis of herbicide resistance, adaptation, and stress resilience in weedy species. Our study directly addresses one of the major bottlenecks identified in that roadmap, by establishing a practical transformation system for cheatgrass. This enables not only functional validation of candidate resistance genes but also broader exploration of the molecular traits that underline rapid weed adaptation. Abbreviations Cauliflower mosaic virus 35S promoter (CaMV 35S), Callus induction medium (CIM), Green fluorescent protein (GFP), Hygromycin phosphotransferase (HPT), Murashige and Skoog (MS) Declarations Ethics declarations Not applicable Funding: The study was supported by the Washington Grain Commission, the R. J. Cook Endowment for Wheat Research, and Pacific Northwest Herbicide Resistance Initiative Authors’ contributions SA and IB designed the study. SA and JM performed the experiments. SA and IB wrote the manuscript. Consent for publications Not applicable Availability of data and materials The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Acknowledgments We thank Dr. Karen Sanguinet for her support in providing access to her fluorescence stereomicroscope for GFP visualization. References Billinton, N., & Knight, A. W. (2001). Seeing the Wood through the Trees: A Review of Techniques for Distinguishing Green Fluorescent Protein from Endogenous Autofluorescence. Analytical Biochemistry , 291 (2), 175–197. https://doi.org/10.1006/abio.2000.5006 Bradley, B. A., Curtis, C. A., Fusco, E. J., Abatzoglou, J. T., Balch, J. 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In Vitro Cellular & Developmental Biology - Plant , 37 (3), 361–368. https://doi.org/10.1007/s11627-001-0064-4 Wong, A. C. S., Massel, K., Lam, Y., Hintzsche, J., & Chauhan, B. S. (2022). Biotechnological Road Map for Innovative Weed Management. Frontiers in Plant Science , 13 , 887723. https://doi.org/10.3389/fpls.2022.887723 Ziemienowicz, A. (2014). Agrobacterium -mediated plant transformation: Factors, applications and recent advances. Biocatalysis and Agricultural Biotechnology , 3 (4), 95–102. https://doi.org/10.1016/j.bcab.2013.10.004 Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 17 Dec, 2025 Reviewers invited by journal 02 Dec, 2025 Editor assigned by journal 19 Nov, 2025 First submitted to journal 17 Nov, 2025 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. 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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-8137674","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":554110502,"identity":"6d9c0f69-bd01-49d5-956a-7dc9693905fc","order_by":0,"name":"Shahbaz Ahmed","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYDACCQY2ZiiDgYGnggHOJlbLGZK18LYRoUV+dvOzxwU1dQz8s7sTH7ydd9je4ADzwds8eLQY3DlmbjzjGBuDxJ2zmw3nbjucuOEAW7I1Xi0SCWbSPGxAJTdyt0nzbrudYHCAByiCz2Ez0r9J8/yTYJC/kbv9N++c20CH8X/Dq4XhRo6ZNG+bAYMB0BZm3obbjBsO8LDh1WJwI6dMemZfAo/hjdzNknOO/U+ceZjN2HIOfodtky74VicndyN344c3NWn2fMebH954g89hUIDkEmYilI+CUTAKRsEowA8AtopKQ5gz55sAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-2273-0779","institution":"Washington State University","correspondingAuthor":true,"prefix":"","firstName":"Shahbaz","middleName":"","lastName":"Ahmed","suffix":""},{"id":554110503,"identity":"173b5837-16b7-48f2-8bea-92e0a220340f","order_by":1,"name":"Jonah Mensonides","email":"","orcid":"","institution":"Washington State University","correspondingAuthor":false,"prefix":"","firstName":"Jonah","middleName":"","lastName":"Mensonides","suffix":""},{"id":554110504,"identity":"4c8de0c6-db0e-4cee-a483-0d64f2f27698","order_by":2,"name":"Ian Burke","email":"","orcid":"","institution":"North Carolina State University","correspondingAuthor":false,"prefix":"","firstName":"Ian","middleName":"","lastName":"Burke","suffix":""}],"badges":[],"createdAt":"2025-11-17 16:31:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8137674/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8137674/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97431452,"identity":"2ce04a1a-dbdc-423d-8f91-d845bcbc574a","added_by":"auto","created_at":"2025-12-04 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10:15:16","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":92257,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8137674/v1/31a62ff51b6761bcc3204fe0.html"},{"id":97431448,"identity":"5b49f948-d60e-42e0-a549-79731ed60351","added_by":"auto","created_at":"2025-12-04 10:15:15","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":307632,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCallus formation from mature and immature embryos.\u003c/strong\u003e (A–B) Soft, non-proliferative tissue forming from mature embryos during the early stages of culture on callus induction medium. (C) Vigorous calli obtained from mature embryos after subculturing. (D) Early callus initiation from immature embryos on callus induction medium. (E) Actively dividing callus derived from immature embryos following subculture.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8137674/v1/8e6966bc2cc34f27a86adf9e.jpeg"},{"id":97431446,"identity":"e7d7d636-5855-4bb8-b375-18827660f377","added_by":"auto","created_at":"2025-12-04 10:15:15","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":352774,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAgrobacterium-mediated transformation of cheatgrass callus. \u003c/strong\u003e(A) Callus immediately after Agrobacteriuminoculation. (B) Callus transferred to selection medium. (C) Hygromycin-resistant callus proliferating on selection medium. (D) Callus observed under brightfield illumination. (E–F) GFP fluorescence in transformed callus under a GFP filter.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8137674/v1/d21c0bbf61c11cac1d2a2a61.jpeg"},{"id":97668351,"identity":"0d2cf002-ad34-45c0-94bc-7ac67762d09e","added_by":"auto","created_at":"2025-12-08 09:25:22","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":276258,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eShoot regeneration and rooting in cheatgrass callus.\u003c/strong\u003e (A–B) Regeneration and rooting of shoots from non-transformed mature embryo-derived callus. (C) Bleached, chlorophyll-deficient shoots from transformed mature embryo-derived callus under hygromycin selection. (D–G) Shoot regeneration and rooting from transformed immature embryo-derived callus, showing successful development of green shoots and roots.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8137674/v1/53d0d6461218956c97da8ad1.jpeg"},{"id":97668275,"identity":"1ee44533-41dc-48d4-82c4-616e4f3968c5","added_by":"auto","created_at":"2025-12-08 09:25:11","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":192341,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGFP visualization in regenerated shoots and mature leaves derived from immature embryo calli.\u003c/strong\u003e (A–D) Regenerated shoots growing on medium imaged under white light and GFP filter. (E–H) Detached mature leaves from regenerated plants observed under brightfield illumination and GFP filter.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8137674/v1/9a29a9752005665f07f46eb8.jpeg"},{"id":97667040,"identity":"bd3bb88d-92f1-4d55-ad50-bb78af97ace6","added_by":"auto","created_at":"2025-12-08 09:22:41","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":116029,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePCR confirmation of transgene integration.\u003c/strong\u003e Amplification of GFP and hygromycin (HPT) fragments in transgenic plants. M=100 bp ladder, T0s = Putative transgenics, P= pGFPGUSPlus plasmid as positive control, WT = DNA from wildtype cheatgrass as negative control, N= No template control. HYG amplicon size= 226 bp, GFP amplicon size = 244.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8137674/v1/24968a45e6499cdb71161dca.jpeg"},{"id":97677646,"identity":"b6bd318e-3f46-4e15-9769-977b7535f637","added_by":"auto","created_at":"2025-12-08 09:53:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2095732,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8137674/v1/cf77a188-ca8a-48a9-aa36-45cb82732fce.pdf"}],"financialInterests":"","formattedTitle":"Establishing a Tissue Culture–based Transformation System for Cheatgrass (Bromus tectorum L.) to Enable Functional Genomic Studies","fulltext":[{"header":"Key Message","content":"\u003cp\u003eThe study establishes the first transformation system for cheatgrass, a highly adaptive and invasive grass, enabling molecular research on herbicide resistance and environmental adaptation.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eWeeds represent one of the greatest challenges to global agriculture, reducing crop yields and increasing production costs across diverse cropping systems (Horvath et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among them, cheatgrass or downy brome (\u003cem\u003eBromus tectorum\u003c/em\u003e L.) is a highly invasive annual grass that has spread extensively across North America and other regions (Bradley et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It dominates rangelands, wheat-based systems, and disturbed habitats, where its success is driven by prolific seed production, seed dormancy, rapid establishment, and tolerance to multiple stresses (Molvar et al., 2024). In addition, the evolution of herbicide resistance, including resistance to glyphosate, further complicates its management (Geddes \u0026amp; Pittman, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Revolinski et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ribeiro, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Despite its ecological and agronomic importance, molecular and genetic studies on cheatgrass remain limited, largely due to the absence of reproducible transformation and regeneration protocols.\u003c/p\u003e\u003cp\u003ePlant transformation technologies have revolutionized crop research by enabling functional gene studies, trait engineering, and genome editing (P. Wang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). While well established for model plants such as \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and major crops such as rice, maize, and wheat (Clough \u0026amp; Bent, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Hayta et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ishida et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Sahoo et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), these approaches have rarely been extended to weedy species. Bringing weeds into the molecular biology laboratory has the potential to transform our understanding of weed biology, herbicide resistance, and adaptation (Wong et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Developing robust tissue culture and transformation protocols for weedy species would open new avenues for dissecting the genetic mechanisms underlying invasiveness and resistance, and for testing biotechnological strategies for weed management (Mellado-S\u0026aacute;nchez et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTissue culture\u0026ndash;based transformation, particularly through \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e\u0026ndash;mediated delivery of transgenes, provides a practical and widely used system for stable genetic modification (Gelvin, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). However, transformation efficiency depends strongly on explant type, callus induction, regeneration capacity, and selection regime (Dhar \u0026amp; Joshi, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Ziemienowicz, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In grasses, particularly non-model species, recalcitrance to transformation has been a persistent challenge. One contributing factor is that \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated gene delivery elicits host immune responses, which can inhibit T-DNA transfer and integration (Luo et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Non-domesticated weed species often exhibit heightened stress resilience and stronger innate defense responses compared with domesticated crops, further reducing their amenability to transformation (Luo et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Mellado-S\u0026aacute;nchez et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e). To date, no transformation system has been reported for cheatgrass.\u003c/p\u003e\u003cp\u003eIn this study, we developed the first \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation protocol for cheatgrass. We optimized callus induction from both mature and immature embryos, evaluated their suitability for transformation, and demonstrated stable green fluorescent protein (GFP) expression in calli and regenerated plants. Molecular confirmation by PCR further validated integration of the transgenes. The work establishes a foundation for molecular genetic studies in cheatgrass and represents an important step toward bringing weedy species into the molecular biology laboratory.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant Material and Seed Preparation\u003c/h2\u003e\u003cp\u003eSeeds of cheatgrass were obtained from a winter-type population maintained under greenhouse conditions, originally collected from Pomeroy, WA. Two types of seed were used: mature seeds and immature seeds. Mature seeds were previously harvested and stored at room temperature in the laboratory. For immature seeds, florets were harvested at early developmental stages before the spikelet turned purple. Seeds were surface sterilized in 10% sodium hypochlorite for 5 min, followed by three rinses with sterile distilled water.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEmbryo Isolation and Callus Induction\u003c/h3\u003e\n\u003cp\u003eSterilized mature seeds were pre-soaked on moistened sterile filter paper for 30 min to facilitate embryo dissection. Embryos were excised under a stereomicroscope in a laminar flow hood and placed scutellum-side down on callus induction medium (CIM). CIM consisted of 4.43 g/L Murashige and Skoog (MS) salts (M519), 30 g/L maltose (M588), and 3.5 g/L Gelzan\u0026trade; (G3251) (all from PhytoTechnology Laboratories, Lenexa, KS, USA). The medium was adjusted to pH 5.8, autoclaved, and allowed to cool to approximately 50\u0026deg;C before adding filter-sterilized supplements: 2.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D; D309), 1.25 mg/L CuSO₄ (C375), 1 g/L casein hydrolysate (C184), and 10 mL/L CI vitamin stock (100 mg/L thiamine HCl (T390), 35 g/L myo-inositol (I703), and 69 g/L L-proline (P698), prepared according to (Hinchliffe \u0026amp; Harwood, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). All components were obtained from PhytoTechnology Laboratories, and the vitamin stock was prepared in advance, filter-sterilized, and stored at 4\u0026deg;C.\u003c/p\u003e\u003cp\u003eImmature embryos were isolated from florets under the microscope at the late milky to early dough stage, when embryos were firm and translucent but before seed coat pigmentation began. Immature embryos were excised using forceps and a teasing needle, then placed scutellum-side down on CIM. Plates were incubated in the dark at 26\u0026ndash;28\u0026deg;C for at least 6\u0026ndash;8 weeks. Actively dividing calli were subcultured every two weeks by fragmentation onto fresh CIM to maintain proliferation.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePlasmid Construction and\u003c/b\u003e \u003cb\u003eAgrobacterium\u003c/b\u003e \u003cb\u003eTransformation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe empty binary vector pGFPGUSPlus ((Plasmid #64401, addgene, Watertown, MA USA), carrying hygromycin resistance, GFP, and GUS reporter genes under the CaMV 35S promoter, was introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101 (rifampicin- and gentamycin-resistant) using a heat-shock method. Competent cells were incubated with 3 \u0026micro;L plasmid DNA on ice for 30 minutes, flash-frozen in liquid nitrogen for 1 min, thawed at 37\u0026deg;C for 2 min, and recovered in 1 mL YEP broth at 28\u0026deg;C for 3 h. Aliquots (50\u0026ndash;150 \u0026micro;L) were plated on YEP agar containing tryptone 10g/L, yeast extract 10g/L, sodium chloride 5g/L, pH7.2, agar 15g/L, 50 mg/L gentamycin and 50 mg/L kanamycin and incubated at 28\u0026deg;C for 48 h.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of\u003c/b\u003e \u003cb\u003eAgrobacterium\u003c/b\u003e \u003cb\u003eCulture for Infection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA single transformed colony was inoculated into 3 mL YEP broth (YEP without agar) with 50 mg/L kanamycin and 50 mg/L gentamycin antibiotics and grown overnight at 28\u0026deg;C with shaking. Glycerol stocks were prepared (700 \u0026micro;L culture\u0026thinsp;+\u0026thinsp;300 \u0026micro;L 50% glycerol) and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. For infection, Cells harboring the pGFPGUSPlus plasmid from glycerol stocks were streaked onto MG/L plates (10 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, 15 g/L agar; pH 7.2) with antibiotics and incubated at 28\u0026deg;C for 24\u0026ndash;48 h. Cells were harvested, resuspended in CIM to an OD₆₀₀ of 0.6, and supplemented with 200 \u0026micro;M acetosyringone.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAgrobacterium\u003c/b\u003e\u003cb\u003e-Mediated Callus Transformation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eActively proliferating calli were immersed in the bacterial suspension for 10 min with occasional shaking, blotted on sterile filter paper, and co-cultivated in the dark at 28\u0026deg;C for 3 days. Calli were then transferred to CIM supplemented with 150 mg/L timentin (for \u003cem\u003eAgrobacterium\u003c/em\u003e elimination) and 40 mg/L hygromycin (for selection) and incubated at 26\u0026ndash;28\u0026deg;C in dark for 2 weeks. After two weeks, the calli was transferred to fresh media for two more weeks with 30 mg/L hygromycin and 150 mg/L timentin.\u003c/p\u003e\n\u003ch3\u003eShoot Regeneration and Rooting\u003c/h3\u003e\n\u003cp\u003eHygromycin-resistant calli were transferred to regeneration medium (4.43 g/L MS salts, 30 g/L maltose, 3.5 g/L Gelzan, 1 mg/L BAP, 0.5 mg/L kinetin, 10 mL/L T/R vitamin stock (Hinchliffe \u0026amp; Harwood, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e), 150 mg/L timentin, and 20 mg/L hygromycin) and incubated at 25\u0026deg;C under a 16 h light/8 h dark photoperiod. Regenerated shoots were transferred to rooting medium for 2\u0026ndash;3 weeks (\u0026frac12;-strength MS salts, 30 g/L maltose, 2g/L Gelzan, 0.5 mg/L indole-3-butyric acid, 15 mg/L hygromycin). Rooted plants were acclimatized in Sunshine Mix 4 (LA4 aggregate) soil in the greenhouse.\u003c/p\u003e\n\u003ch3\u003eGFP Fluorescence Screening\u003c/h3\u003e\n\u003cp\u003eGFP signals in calli and regenerated plants were detected using a Leica MDG41 fluorescence stereomicroscope. Samples were first observed under brightfield illumination to assess tissue morphology and then re-examined using a GFP filter set (excitation 450\u0026ndash;490 nm; emission 500\u0026ndash;550 nm). To minimize the possibility of misinterpreting autofluorescence, imaging was performed under complete darkness, and tissues were examined sequentially under multiple filters sets such as white light, UV and mCherry to determine if the signal is present under all filters or just GFP. For regenerated plants, detached leaves were placed on glass slides and visualized under the same filter conditions. GFP-positive tissues were identified by distinct green fluorescence, which was documented using the integrated Leica digital camera system.\u003c/p\u003e\n\u003ch3\u003eGenomic DNA Extraction and PCR Confirmation\u003c/h3\u003e\n\u003cp\u003eLeaf tissue (1\u0026ndash;2 cm) from GFP-positive putative T0 at four leaves stages growing in soil was collected, and genomic DNA was extracted using the MagMax DNA Extraction Kit (Thermo Fisher Scientific) following the manufacturer\u0026rsquo;s instructions on the KingFisher Apex (Thermo Fisher Scientific) automated system.\u003c/p\u003e\u003cp\u003ePCR amplification was performed using GreenDream Taq PCR Master Mix (Thermo Fisher Scientific) with primer pairs designed to amplify two transgene regions: a 244 bp fragment spanning the CaMV 35S promoter and GFP junction, and a 226 bp fragment spanning the CaMV 35S promoter and the hygromycin phosphotransferase (HPT) gene. Wild-type genomic DNA and plasmid DNA were included as negative and positive controls, respectively, alongside a no-template control.\u003c/p\u003e\u003cp\u003eAmplification was performed on a Bio-Rad T100 thermal cycler under the following program: initial denaturation at 95\u0026deg;C for 3 min; 34 cycles of 95\u0026deg;C for 30 s, 58\u0026deg;C for 30 s, and 72\u0026deg;C for 30 s; and a final extension at 72\u0026deg;C for 10 min. PCR products were resolved on 2% agarose gels stained with SYBR Safe DNA stain (Thermo Fisher Scientific) and visualized under UV illumination.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eCallus formation from Mature and Immature Embryos\u003c/h2\u003e\u003cp\u003eBoth mature and immature embryos were evaluated as explant sources. In mature embryos, callus formation occurred in two distinct phases. During the initial phase, a soft, non-proliferative tissue developed within two weeks, with no further growth observed over the following 4\u0026ndash;6 weeks. Subsequently, actively dividing calli were initiated, which required an additional 2\u0026ndash;3 weeks to attain sufficient size and vigor. Upon subculturing, these proliferating calli exhibited a 100% regrowth rate. Overall, approximately 15\u0026ndash;20% of mature embryos successfully generated actively growing calli (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCallus formation from immature embryos was faster, with actively proliferating calli observed from the beginning of the culture period, and full-sized calli obtained within 6\u0026ndash;8 weeks. However, the efficiency of healthy callus induction was lower, with less than 10% of immature embryos producing viable calli (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Once established, these calli maintained 100% growth upon subculture and provided abundant material for subsequent regeneration and transformation experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAgrobacterium\u003c/b\u003e\u003cb\u003e-mediated Transformation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBoth mature- and immature-derived calli were amenable to \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e-mediated transformation using a binary vector carrying \u003cem\u003eGFP\u003c/em\u003e and a hygromycin resistance marker. Stable GFP fluorescence was detected in sectors of hygromycin-resistant calli derived from both types of embryos and persisted after subculturing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-F).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRegeneration of Transgenic Plants\u003c/h3\u003e\n\u003cp\u003eTo establish baseline regeneration efficiency, non-transformed calli derived from both mature and immature embryos were first evaluated. Under regeneration conditions, up to 50% of healthy, proliferating calli from mature and immature embryo regenerated into green, growing shoots (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). These shoots readily produced roots in rooting medium and acclimatized successfully to soil in the greenhouse (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHowever, calli derived from mature embryos exhibited a sharp decline in regeneration efficiency on selection media following \u003cem\u003eAgrobacterium\u003c/em\u003e transformation, yielding primarily bleached, chlorophyll-deficient shoots under hygromycin selection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) In comparison, immature embryo-derived calli post \u003cem\u003eAgrobacterium\u003c/em\u003e transformation showed superior regeneration potential. Under selection, small greening spots appeared on immature embryo derived calli, which gradually developed into shoots that elongated and remained healthy on hygromycin-containing medium. These shoots subsequently formed strong root systems on rooting medium and acclimatized successfully when transferred to soil (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-G).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMolecular Confirmation of Transgene Integration\u003c/h2\u003e\u003cp\u003eRegenerated shoots exhibited distinct localized GFP fluorescence signals that were absent in non-transgenic controls, and fluorescence remained detectable in the leaves of fully regenerated plants, confirming transgene expression in mature tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;H).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAmplification through PCR of junction fragments between the CaMV 35S promoter and either the \u003cem\u003eGFP\u003c/em\u003e or hygromycin phosphotransferase (HPT) gene yielded the expected amplicons in all putative transgenic lines. No products were detected in wild-type or no-template controls, whereas plasmid DNA produced the expected fragments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe study presents the first successful protocol for \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated genetic transformation of cheatgrass, a highly invasive and agriculturally significant weed. By combining embryo-derived callus induction, optimized selection regimes, and regeneration assays, we demonstrate that cheatgrass is amenable to tissue culture\u0026ndash;based transformation, thereby establishing a platform for molecular genetic studies in this species.\u003c/p\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eCallus induction, and agrobacterium transformation\u003c/h2\u003e\u003cp\u003eWe detect clear differences in callus induction and regeneration between mature and immature embryos. In mature embryos, the initial soft tissue stage represents limited dedifferentiation before a subset of responsive cells re-enters the cell cycle and generates actively dividing calli. Similar to the results reported by (Nakasha et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), increasing 2,4-D concentration in our study did not enhance callus formation in mature embryos (data not shown). This is likely because mature embryos are composed of differentiated, less mitotically active cells, which are slower to respond to auxin-rich induction media (Ganeshan et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Suo et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Once established, however, subculture of actively growing calli from mature embryos consistently generates sufficient material for downstream applications, including transformation and shoot regeneration.\u003c/p\u003e\u003cp\u003eImmature embryos, excised from florets before visible purple pigmentation, initiate actively dividing calli from the start but at lower frequency. This reduction is due to several factors. First, florets at very early stages are watery, making excision technically difficult and increasing the likelihood of mechanical damage. Second, embryo size is small compared with other crops such as maize, wheat, or \u003cem\u003eBrachypodium\u003c/em\u003e, and slight variation in developmental stage introduces heterogeneity in response. Similar trade-offs between embryo developmental stage, callus induction frequency, and regeneration potential have been reported in cereals such as barley and wheat (Hinchliffe \u0026amp; Harwood, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Ishida et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Despite the lower initial frequency, once actively growing calli is established from immature embryos, subculture produces fresh, proliferating callus at nearly 100% efficiency. Continuous subculturing provides ample material for transformation experiments.\u003c/p\u003e\u003cp\u003eOnce actively growing calli was obtained, both mature-derived and immature-derived tissues were amenable to \u003cem\u003eAgrobacterium\u003c/em\u003e transformation. Putative transgenic calli maintained active growth and were readily identified under GFP filters. Autofluorescence is common in grass tissues due to chlorophyll and phenolic compounds, and most calli display a dull green background under blue light (Billinton \u0026amp; Knight, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Donaldson, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, true GFP-positive calli emit strong, localized signals that are readily distinguished from background when samples are observed first under brightfield illumination, mcherry and then under GFP filters in complete darkness (Mellado-S\u0026aacute;nchez et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e). The persistence of GFP signals through multiple rounds of subculture provides strong evidence for stable T-DNA integration.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eSelection and regeneration bottlenecks\u003c/h2\u003e\u003cp\u003eA major bottleneck of this system is the reduced transformation efficiency and regeneration capacity of transgenic calli. Non-transformed calli regenerate green shoots at relatively high frequency, whereas only a small fraction of hygromycin-resistant calli from immature embryos produce viable shoots due to selection indicating lower transformation efficiency. The lower transformation efficiency in non-domesticated plant species is due to their increased immunity against biotic and abiotic stresses (Luo et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Singh \u0026amp; van der Knaap, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Calli derived from mature embryos regenerated even less efficiently post \u003cem\u003eagrobacterium\u003c/em\u003e transformation (Ward \u0026amp; Jordan, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), often producing bleached, chlorophyll-deficient shoots. In our system, the duration of culture may be particularly critical: mature embryos require 6\u0026ndash;8 weeks to produce actively dividing calli, followed by additional rounds of subculture to generate sufficient material, before transformation and selection steps are imposed. As a result, regenerated shoots from mature embryo calli are derived from tissues that have already spent 10\u0026ndash;12 weeks on callus induction medium, potentially reducing regenerative capacity. By the time transformed calli are transferred to regeneration medium, the tissue may be too old to support the development of green shoots (Chakravarty \u0026amp; Goswami, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Orshinsky \u0026amp; Tomes, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1985\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn immature embryo-derived calli, the shorter culture duration before transformation likely contributes to more successful regeneration into green shoots. However, we also observe that shoots emerging from immature calli must remain in direct contact with regeneration medium; those that grow above the surface eventually senesce (Mazumdar et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This problem can be alleviated by frequent transfer to fresh medium, ensuring that developing shoots maintain contact. Root formation occurs more efficiently when regenerated shoots are transferred to a reduced selective rooting medium, a strategy consistent with protocols in barley and rice where selective pressure is relaxed during late developmental stages (Hayta et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Transplanted plantlets establish well in soil, producing fertile T0 plants.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe ability to stably transform cheatgrass has significant implications for weed science. Weeds remain largely excluded from the molecular biology toolkit, despite their major role in agricultural yield loss and herbicide resistance evolution. As emphasized in the biotechnological roadmap for weed management (Wong et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), transformation and functional genomics are prerequisite tools for dissecting the genetic basis of herbicide resistance, adaptation, and stress resilience in weedy species. Our study directly addresses one of the major bottlenecks identified in that roadmap, by establishing a practical transformation system for cheatgrass. This enables not only functional validation of candidate resistance genes but also broader exploration of the molecular traits that underline rapid weed adaptation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCauliflower mosaic virus 35S promoter (CaMV 35S), Callus induction medium (CIM), Green fluorescent protein (GFP), Hygromycin phosphotransferase (HPT), Murashige and Skoog (MS)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was supported by the Washington Grain Commission, the R. J. Cook Endowment for Wheat Research, and Pacific Northwest Herbicide Resistance Initiative\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSA and IB designed the study.\u003c/p\u003e\n\u003cp\u003eSA and JM performed the experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSA and IB wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publications\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Karen Sanguinet for her support in providing access to her fluorescence stereomicroscope for GFP visualization.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eBillinton, N., \u0026amp; Knight, A. 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Springer New York. https://doi.org/10.1007/978-1-4939-8944-7_8\u003c/p\u003e\n\u003cp\u003eHinchliffe, A., \u0026amp; Harwood, W. A. (2019b). Agrobacterium-Mediated Transformation of Barley Immature Embryos. In W. A. Harwood (Ed.), \u003cem\u003eBarley\u003c/em\u003e (Vol. 1900, pp. 115\u0026ndash;126). Springer New York. https://doi.org/10.1007/978-1-4939-8944-7_8\u003c/p\u003e\n\u003cp\u003eHorvath, D. P., Clay, S. A., Swanton, C. J., Anderson, J. V., \u0026amp; Chao, W. S. (2023). Weed-induced crop yield loss: A new paradigm and new challenges. \u003cem\u003eTrends in Plant Science\u003c/em\u003e, \u003cem\u003e28\u003c/em\u003e(5), 567\u0026ndash;582. https://doi.org/10.1016/j.tplants.2022.12.014\u003c/p\u003e\n\u003cp\u003eIshida, Y., Hiei, Y., \u0026amp; Komari, T. (2007). 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(2014). \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated plant transformation: Factors, applications and recent advances. \u003cem\u003eBiocatalysis and Agricultural Biotechnology\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e(4), 95\u0026ndash;102. https://doi.org/10.1016/j.bcab.2013.10.004\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bromus tectorum, cheatgrass, Downy brome, Agrobacterium tumefaciens, tissue culture, GFP, herbicide resistance, molecular weed biology","lastPublishedDoi":"10.21203/rs.3.rs-8137674/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8137674/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCheatgrass (\u003cem\u003eBromus tectorum\u003c/em\u003e L.) is one of the most invasive annual grasses in the western United States. It has spread extensively across rangelands and cropping systems and increasingly expresses herbicide resistance. Despite its ecological and agricultural significance as a major invasive weed, functional genetic studies in cheatgrass have been hindered by the absence of a reproducible transformation system. Here, we report the first successful protocol for \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e\u0026ndash;mediated genetic transformation of cheatgrass. Mature and immature embryos were evaluated as explant sources to optimize callus induction and regeneration. Callus formation from immature embryos began earlier than from mature embryos, which required a longer culture period. Hygromycin-resistant calli expressing green fluorescent protein (GFP) were obtained from both explant types, confirming successful genetic transformation. Following \u003cem\u003eAgrobacterium\u003c/em\u003e transformation, calli derived from immature embryos regenerated green, viable shoots, whereas those from mature embryos frequently produced bleached, chlorophyll-deficient tissues. PCR amplification of \u003cem\u003eGFP\u003c/em\u003e and \u003cem\u003ehygromycin phosphotransferase (HPT)\u003c/em\u003e confirmed stable integration of transgenes into the cheatgrass genome. Our work presents the first reproducible immature embryo\u0026ndash;based transformation system for cheatgrass, enabling molecular genetics and functional genomics studies in weedy grasses.\u003c/p\u003e","manuscriptTitle":"Establishing a Tissue Culture–based Transformation System for Cheatgrass (Bromus tectorum L.) to Enable Functional Genomic Studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 10:15:11","doi":"10.21203/rs.3.rs-8137674/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-12-17T05:33:09+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-02T13:57:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-19T06:26:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2025-11-17T11:29:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e9d26cf1-f375-4fef-86b2-82efdedb78af","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-04T10:15:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-04 10:15:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8137674","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8137674","identity":"rs-8137674","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}