Determining the genome content of ornamental plants using flow cytometry

Determining the genome content of ornamental plants using flow cytometry | 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 Short Report Determining the genome content of ornamental plants using flow cytometry Harini Srikanth Karumbati, Raksha K K, Sarvajith M, Krishnamurthy H This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9111048/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Genome size is a key genomic trait influencing plant growth, development, and breeding potential. Here, nuclear DNA content of six commonly cultivated ornamental plant species was estimated using flow cytometry using propidium iodide staining. Nuclei were isolated from young leaf tissues and stained with propidium iodide (PI). Chicken red blood cells served as an internal reference standard for genome size calculation. All samples produced high-quality DNA histograms with coefficients of variation below 5%. Genome size considerably varied among the studies species, ranging from 0.302 pg in 0.302 pg in Alocasia wentii to 2.461 pg in Pilea cadierei . Intermediate genome sizes were observed in Clerodendrum chinense , Codiaeum variegatum , Spathiphyllum kochii , and Calathea zebrina . The observed variation suggests genomic diversity in ornamental plants and demonstrates the flow cytometry as a tool, for rapid estimation of genome size. Flow cytometry Ornamental plants Genome size Nuclear DNA content Plant breeding Figures Figure 1 Figure 2 Figure 3 1. Introduction Genome size defined as the amount of nuclear DNA within a cell is a highly variable feature of plant genomes. Among angiosperms, genome size differs by several orders of magnitude and has been linked to biological traits such as cell size, growth rate and ecological adaptability among others. The variation reflects differences in genome organisation and provides insights into plant diversity and evolution (Leitch et al., 2019 ). Despite its importance, we still lack information regarding many cultivated ornamental plants. Flow cytometry has gained importance because it allows rapid, precise and reproducible measurement of nuclear DNA content from limited sample volume. With appropriate internal reference standards and optimised protocols, flow cytometry enables rapid and reliable comparison of genomes across species (Doležel et al., 2005; Basak et al., 2018 ; Nix et al., 2024 ). Ornamental plants constitute taxonomically and morphologically diverse group that is of significant horticulture and economic value. Genome size variations within the ornamental species can provide useful information for evolutionary comparisons and hybridisation strategies (van de Peer et al., 2021 ). Despite its relevance, genome size information remains incomplete for many cultivated ornamental plants. Although genome size has been reported for some ornamental taxa, current research on ornamental plants is mainly focused on methods or techniques for improving propagation, production, and postharvest or postproduction handling. Given the diversity and multipurpose use of ornamental plants, along with their increasing production worldwide, require more fundamental research on their growth and development, flower and leaf colour, fragrance, and growth form (Chen et al., 2021). Therefore, the present study is aimed to estimate the nuclear DNA content of six ornamental plant species using flow cytometry and to assess interspecific variation in genome size. 2. Materials and Methods 2.1 Plant Materials Young and healthy leaf tissues were collected from six commonly cultivated ornamental plants from two locations: the herbal garden at the National Centre for Biological Sciences (NCBS) and the nursery at Institute of Stem Cell Science and Regenerative Medicine (inStem), Bangalore, India (Fig. 1 ). A total of six plant species were collected (in minimum three replicates). Alocasia wentii, Clerodendrum chinense, Pilea cadierei, Codiaeum variegatum, Spathiphyllum kochii, Calathea zebrina plants were collected. These plants were maintained under normal growth conditions, and fresh fully expanded young leaves were selected to ensure high nuclear integrity and minimal accumulation of secondary metabolites (Table 1 ). Table 1 Genome size of various plants species studied in the current study Common Name Sample Origin Mean Channel Number Genome Content (pg) Mbps* New Guinea Shield Alocasia wentii New Guinea 6.539 0.302 295.356 Madras Mallige Clerodendrum chinense Nepal, the Himalayas, Assam, the Andaman and Nicobar Islands 17.447 0.687–0.920 (0.804) 786.312 Aluminium Plant Pilea cadierei Vietnam 53.791 2.461 2406.858 Garden Croton Codiaeum variegatum Malaysia and the Pacific 14.603 0.676 661.128 Peace Lily Spathiphyllum kochii Tropical regions of the Americas and south-eastern Asia. 35.562 1.744–2.183 (1.964) 1920.792 Zebra Plant Calathea zebrina South-eastern Brazil. 20.467 0.584–0.998 (0.791) 773.598 1pg = 978 Mbps 2.2 Sample preparation Nuclei from the plant cells were isolated using Nuclear Isolation Buffer (NIB) containing 50 µg/ml propidium iodide (PI) prepared in in 1 g/l trisodium citrate dihydrate supplemented with 0.05% (v/v) of non-ionic detergent (Igepal CA-630), 2 mg/ml RNase A, 1% (w/v) PVP - Polyvinylpyrrolidone. Briefly, 2 ml od NIB was dispensed into sterile petri plate and two to three young leaf tips (approximately 0.5 inch in length) were placed in the buffer. The leaf tissue was finely and vigorously chopped using surgical blade. The resulting suspension, including the residual leaf fragments was transferred to a 5 ml tube and gently agitated by repeated pipetting using a dropper to ensure thorough mixing. The homogenate was subsequently filtered through 30 µm nylon mesh to remove debris. The resulting clean suspension was used for further analysis. 2.3 Microscopy After extraction, sample suspension was observed in bright-field microscope to assess the presence of intact nuclei and cellular debris. 2.4 Flow cytometric analysis PI-stained nuclear samples were analysed using benchtop FACS Fortessa X-20 (BD Biosciences). The PI fluorescence was excited using 561 nm solid state LASER and the emission was collected using 585/20 band pass filter. Prior to sample acquisition, instrument performance and fluorescent linearity was verified using DNA QC kit (BD Biosciences) following manufacturer instructions. For each sample, frequency distribution of histograms of PI-stained nuclei were generated and used for data analysis. Only histograms exhibiting coefficient of variation (CV) less than 5% for the G 0 /G 1 signal were considered acceptable for genome size estimation. Chicken blood cells (CRBC) with known DNA content of 2.3 pg per diploid nucleus (Yang et al., 2019) were used as internal reference standard for genome size calculation. The nuclear DNA content of the samples was estimated in picogram using the following formula relative to CRBC standard (Doležel et al., 2007 ). \(\text{D}\text{N}\text{A}\text{c}\text{o}\text{n}\text{t}\text{e}\text{n}\text{t}\left(\text{p}\text{g}\right)=\frac{\text{C}\text{R}\text{B}\text{C}\text{D}\text{N}\text{A}\text{c}\text{o}\text{n}\text{t}\text{e}\text{n}\text{t}\times\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\text{m}\text{e}\text{a}\text{n}\text{f}\text{l}\text{u}\text{o}\text{r}\text{e}\text{s}\text{c}\text{e}\text{n}\text{c}\text{e}\text{c}\text{h}\text{a}\text{n}\text{n}\text{e}\text{l}\text{n}\text{o}.}{\text{C}\text{R}\text{B}\text{C}\text{m}\text{e}\text{a}\text{n}\text{f}\text{l}\text{u}\text{o}\text{r}\text{e}\text{s}\text{c}\text{e}\text{n}\text{c}\text{e}\text{c}\text{h}\text{a}\text{n}\text{n}\text{e}\text{l}\text{n}\text{o}.}\) 3. Results and discussion Genome size estimation of six ornamental plant species was carried out using flow cytometry with CRBC as an internal reference standard (2C = 2.30 pg). Initial bright-field microscopic examination confirmed the presence of intact nuclei with minimal debris (Fig. 2 ). Flow cytometric analysis of extract showed a clear and well resolved DNA histograms with coefficients of variation below 5%. The estimated nuclear DNA content of the studied ornamental plants is summarized in Table 1 . A wide range of genome sizes was observed among the species analyzed. The smallest genome size was in Alocasia wentii (New Guinea Shield), with a DNA content of 0.302 pg (approximately 295 Mbp), whereas Pilea cadierei (Aluminium Plant) exhibited the largest genome size of 2.461 pg (approximately 2407 Mbp). Intermediate genome sizes were observed in Clerodendrum chinense , Codiaeum variegatum , Spathiphyllum kochii , and Calathea zebrina , with DNA contents ranging from approximately 0.676 to 1.964 pg (Fig. 3 ). The results demonstrate substantial interspecific variation in genome size among commonly cultivated ornamental plants. Such variation in nuclear DNA content is a well-recognized feature of angiosperms and reflects differences in genome organization, repetitive DNA content, and evolutionary history (Sliwinska et al., 2018). Genome size has been linked to influence several biological traits, including cell size, growth rate, and developmental timing, which may indirectly affect ornamental characteristics such as leaf size, plant architecture, and flowering behaviour. In this study, PI staining was employed for genome size estimation due to its stoichiometric binding to double-stranded DNA. In contrast, DAPI may introduce bias associated to base composition particularly differences in GC/AT content (Basak et al., 2018 ; Nix et al., 2024 ). From a breeding perspective, nuclear DNA content can support the selection of compatible parental lines, assist in planning hybridization strategies, and support approaches involving polyploidy for trait improvement. The observed genome size diversity among the studied species suggests that the ornamental plants are genetically diverse group with considerable scope for breeding and improvement. Additionally, genome size estimation using flow cytometry is rapid and easy (Nix et al., 2024 ). Future studies can integrate flow cytometry with complementary cytogenetic and molecular approaches such as chromosome analysis, ploidy determination, and genome sequencing to gain deeper insights into understand genome structure, organisation and evolutionary relationship among ornamental plants (Zheng et al., 2021 ; Kishi-Kaboshi et al., 2018 ). Declarations Conflict of interest None Author Contribution HSK: Analysed, data curation, Manuscript- writing original RK: Analysis, data curationSM: Manuscript- writing original; review editingHK : Supervision, Manuscript- review editing Acknowledgement The authors acknowledge the Central Imaging & Flow Cytometry of National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bengaluru. References Leitch, I. J, Johnston E, Pellicer J, Hidalgo O, Bennett MD. 2019. Angiosperm DNA C-values database (release 9.0, Apr 2019) https://cvalues.science.kew.org/ Doležel, J., Bartoš, J. A. N. Plant DNA flow cytometry and estimation of nuclear genome size. Ann. Bot. 2005, 95(1), 99–110. Basak, S., Krishnamurthy, H., Rangan, L., Genome size variation among 3 selected genera of Zingiberoideae. Meta Gene 2018, 15, 42–49. Nix, J., Ranney, T. G., Lynch, N. P., Chen, H., Flow cytometry for estimating plant genome size: revisiting assumptions, sources of variation, reference standards, and best practices. J. Am. Soc. Hortic. Sci. 2024, 149(3), 131–141. Van de Peer, Y., Ashman, T. L., Soltis, P. S., Soltis, D. E., Polyploidy: an evolutionary and ecological force in stressful times. Plant Cell, 2021, 33(1), 11–26. Chen, J., Ornamental plant research inaugural editorial. Ornamental Plant Research, 2021, 1(1), 1–2. Doležel, J., Greilhuber, J., Suda, J., Estimation of nuclear DNA content in plants using flow cytometry. Nat. Protoc. 2007, 2(9), 2233–2244. Sliwinska, E., Flow cytometry-a modern method for exploring genome size and nuclear DNA synthesis in horticultural and medicinal plant species. Folia Hortic. 2018, 30(1), 103. Zheng, T., Li, P., Li, L., Zhang, Q., Research advances in and prospects of ornamental plant genomics. Hortic. Res. 2021, 8(1). Kishi-Kaboshi, M., Aida, R., Sasaki, K., Genome engineering in ornamental plants: current status and future prospects. Plant Physiol. Biochem. 2018, 131, 47–52. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-9111048","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":620430678,"identity":"a8825436-388d-4991-8ff7-bbca7b245918","order_by":0,"name":"Harini Srikanth Karumbati","email":"","orcid":"","institution":"Vidya Niketan School","correspondingAuthor":false,"prefix":"","firstName":"Harini","middleName":"Srikanth","lastName":"Karumbati","suffix":""},{"id":620430679,"identity":"79c5853c-c2e1-420e-8939-97898b3a891a","order_by":1,"name":"Raksha K K","email":"","orcid":"","institution":"National Centre for Biological Sciences","correspondingAuthor":false,"prefix":"","firstName":"Raksha","middleName":"K","lastName":"K","suffix":""},{"id":620430680,"identity":"b4e67e73-0ef3-417b-8013-44ec2a02b486","order_by":2,"name":"Sarvajith M","email":"","orcid":"","institution":"National Centre for Biological Sciences","correspondingAuthor":false,"prefix":"","firstName":"Sarvajith","middleName":"","lastName":"M","suffix":""},{"id":620430681,"identity":"cfe2d382-4705-4e1f-a0c8-86316d4a23a0","order_by":3,"name":"Krishnamurthy H","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYBACCQbGBmYgLcfAwAMVYiZSizEpWiBqEhvgWggBydmHmz8X7rBL385+9vAHxj029gzsvAfwapHmS2yTnnkmOXdnT16aBMOztMQGZr4EvFrkeBjbmHnbmHM33OAxY2A4cDiBgZnHgJCW5s+8bfXpBjd4jD8wHPhvT1CLNA9jgzRv2+EEoBYDCYYDB4ABSECLZA9jG1DLccMNZ4B+STiQnNhGSIvEGfbHQIdVyxscB4bYhwN29vz8Z/BrQQUJQMxGgvpRMApGwSgYBTgAAGS3Ox8ir5n5AAAAAElFTkSuQmCC","orcid":"","institution":"National Centre for Biological Sciences","correspondingAuthor":true,"prefix":"","firstName":"Krishnamurthy","middleName":"","lastName":"H","suffix":""}],"badges":[],"createdAt":"2026-03-13 06:39:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9111048/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9111048/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106760421,"identity":"a1aa4d0f-50cc-4d5a-8212-f0d6b73753ca","added_by":"auto","created_at":"2026-04-13 08:44:33","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":56459,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of ornamental plants used in this study. \u003cem\u003eClerodendrum chinense\u003c/em\u003e (Madras mallige) (A), \u003cem\u003ePilea cadierei\u003c/em\u003e(Aluminium plant) (B), \u003cem\u003eSpathiphyllum kochii\u003c/em\u003e (Peace lily) (C) and \u003cem\u003eCodiaeum variegatum\u003c/em\u003e (Garden croton) (D).\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9111048/v1/d9e357d68881c9ac47545aad.jpg"},{"id":106760425,"identity":"e1145fe5-b403-4fd3-9eac-805cf518018e","added_by":"auto","created_at":"2026-04-13 08:44:36","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":11712,"visible":true,"origin":"","legend":"\u003cp\u003eBright filed view of nuclei extracted from \u003cem\u003eClerodendrum chinense\u003c/em\u003e, commonly known as Madras Mallige.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9111048/v1/b02dde2a4e521b87a0ba5ce4.jpg"},{"id":106760382,"identity":"4717708b-84c9-435d-b538-65f553eb502f","added_by":"auto","created_at":"2026-04-13 08:44:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":57268,"visible":true,"origin":"","legend":"\u003cp\u003eHistogram of fluorescence intensity of CRBC nuclei (A), \u003cem\u003ePilea cadierei\u003c/em\u003e (Aluminium plant) (B), \u003cem\u003eClerodendrum chinense\u003c/em\u003e (Madras mallige) (C) stained with hypotonic PI (A).\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9111048/v1/dc1e192d5b946e30908969d2.jpg"},{"id":106760442,"identity":"d1f216a3-f3e2-4396-8394-4e0f89442eb0","added_by":"auto","created_at":"2026-04-13 08:44:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":528758,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9111048/v1/ba3975ac-ce35-48bf-8997-6ec21b61d058.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Determining the genome content of ornamental plants using flow cytometry","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGenome size defined as the amount of nuclear DNA within a cell is a highly variable feature of plant genomes. Among angiosperms, genome size differs by several orders of magnitude and has been linked to biological traits such as cell size, growth rate and ecological adaptability among others. The variation reflects differences in genome organisation and provides insights into plant diversity and evolution (Leitch et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Despite its importance, we still lack information regarding many cultivated ornamental plants.\u003c/p\u003e \u003cp\u003eFlow cytometry has gained importance because it allows rapid, precise and reproducible measurement of nuclear DNA content from limited sample volume. With appropriate internal reference standards and optimised protocols, flow cytometry enables rapid and reliable comparison of genomes across species (Doležel et al., 2005; Basak et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nix et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOrnamental plants constitute taxonomically and morphologically diverse group that is of significant horticulture and economic value. Genome size variations within the ornamental species can provide useful information for evolutionary comparisons and hybridisation strategies (van de Peer et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Despite its relevance, genome size information remains incomplete for many cultivated ornamental plants.\u003c/p\u003e \u003cp\u003eAlthough genome size has been reported for some ornamental taxa, current research on ornamental plants is mainly focused on methods or techniques for improving propagation, production, and postharvest or postproduction handling. Given the diversity and multipurpose use of ornamental plants, along with their increasing production worldwide, require more fundamental research on their growth and development, flower and leaf colour, fragrance, and growth form (Chen et al., 2021). Therefore, the present study is aimed to estimate the nuclear DNA content of six ornamental plant species using flow cytometry and to assess interspecific variation in genome size.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant Materials\u003c/h2\u003e \u003cp\u003eYoung and healthy leaf tissues were collected from six commonly cultivated ornamental plants from two locations: the herbal garden at the National Centre for Biological Sciences (NCBS) and the nursery at Institute of Stem Cell Science and Regenerative Medicine (inStem), Bangalore, India (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A total of six plant species were collected (in minimum three replicates). \u003cem\u003eAlocasia wentii, Clerodendrum chinense, Pilea cadierei, Codiaeum variegatum, Spathiphyllum kochii, Calathea zebrina\u003c/em\u003e plants were collected. These plants were maintained under normal growth conditions, and fresh fully expanded young leaves were selected to ensure high nuclear integrity and minimal accumulation of secondary metabolites (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenome size of various plants species studied in the current study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCommon Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOrigin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003cp\u003eChannel\u003c/p\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGenome\u003c/p\u003e \u003cp\u003eContent (pg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMbps*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNew Guinea\u003c/p\u003e \u003cp\u003eShield\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAlocasia wentii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNew Guinea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.539\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.302\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e295.356\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMadras Mallige\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eClerodendrum chinense\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNepal, the Himalayas, Assam, the Andaman and Nicobar Islands\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17.447\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.687\u0026ndash;0.920\u003c/p\u003e \u003cp\u003e(0.804)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e786.312\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAluminium\u003c/p\u003e \u003cp\u003ePlant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePilea cadierei\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVietnam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e53.791\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.461\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2406.858\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGarden Croton\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCodiaeum variegatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMalaysia and the Pacific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14.603\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.676\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e661.128\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePeace Lily\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSpathiphyllum kochii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTropical regions of the Americas and south-eastern Asia.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e35.562\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.744\u0026ndash;2.183\u003c/p\u003e \u003cp\u003e(1.964)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1920.792\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZebra Plant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCalathea zebrina\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSouth-eastern Brazil.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20.467\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.584\u0026ndash;0.998\u003c/p\u003e \u003cp\u003e(0.791)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e773.598\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e1pg\u0026thinsp;=\u0026thinsp;978 Mbps\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Sample preparation\u003c/h2\u003e \u003cp\u003eNuclei from the plant cells were isolated using Nuclear Isolation Buffer (NIB) containing 50 \u0026micro;g/ml propidium iodide (PI) prepared in in 1 g/l trisodium citrate dihydrate supplemented with 0.05% (v/v) of non-ionic detergent (Igepal CA-630), 2 mg/ml RNase A, 1% (w/v) PVP - Polyvinylpyrrolidone.\u003c/p\u003e \u003cp\u003eBriefly, 2 ml od NIB was dispensed into sterile petri plate and two to three young leaf tips (approximately 0.5 inch in length) were placed in the buffer. The leaf tissue was finely and vigorously chopped using surgical blade. The resulting suspension, including the residual leaf fragments was transferred to a 5 ml tube and gently agitated by repeated pipetting using a dropper to ensure thorough mixing. The homogenate was subsequently filtered through 30 \u0026micro;m nylon mesh to remove debris. The resulting clean suspension was used for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Microscopy\u003c/h2\u003e \u003cp\u003eAfter extraction, sample suspension was observed in bright-field microscope to assess the presence of intact nuclei and cellular debris.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Flow cytometric analysis\u003c/h2\u003e \u003cp\u003ePI-stained nuclear samples were analysed using benchtop FACS Fortessa X-20 (BD Biosciences). The PI fluorescence was excited using 561 nm solid state LASER and the emission was collected using 585/20 band pass filter. Prior to sample acquisition, instrument performance and fluorescent linearity was verified using DNA QC kit (BD Biosciences) following manufacturer instructions. For each sample, frequency distribution of histograms of PI-stained nuclei were generated and used for data analysis. Only histograms exhibiting coefficient of variation (CV) less than 5% for the G\u003csub\u003e0\u003c/sub\u003e/G\u003csub\u003e1\u003c/sub\u003e signal were considered acceptable for genome size estimation. Chicken blood cells (CRBC) with known DNA content of 2.3 pg per diploid nucleus (Yang et al., 2019) were used as internal reference standard for genome size calculation. The nuclear DNA content of the samples was estimated in picogram using the following formula relative to CRBC standard (Doležel et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\text{D}\\text{N}\\text{A}\\text{c}\\text{o}\\text{n}\\text{t}\\text{e}\\text{n}\\text{t}\\left(\\text{p}\\text{g}\\right)=\\frac{\\text{C}\\text{R}\\text{B}\\text{C}\\text{D}\\text{N}\\text{A}\\text{c}\\text{o}\\text{n}\\text{t}\\text{e}\\text{n}\\text{t}\\times\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}\\text{m}\\text{e}\\text{a}\\text{n}\\text{f}\\text{l}\\text{u}\\text{o}\\text{r}\\text{e}\\text{s}\\text{c}\\text{e}\\text{n}\\text{c}\\text{e}\\text{c}\\text{h}\\text{a}\\text{n}\\text{n}\\text{e}\\text{l}\\text{n}\\text{o}.}{\\text{C}\\text{R}\\text{B}\\text{C}\\text{m}\\text{e}\\text{a}\\text{n}\\text{f}\\text{l}\\text{u}\\text{o}\\text{r}\\text{e}\\text{s}\\text{c}\\text{e}\\text{n}\\text{c}\\text{e}\\text{c}\\text{h}\\text{a}\\text{n}\\text{n}\\text{e}\\text{l}\\text{n}\\text{o}.}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003eGenome size estimation of six ornamental plant species was carried out using flow cytometry with CRBC as an internal reference standard (2C\u0026thinsp;=\u0026thinsp;2.30 pg). Initial bright-field microscopic examination confirmed the presence of intact nuclei with minimal debris (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Flow cytometric analysis of extract showed a clear and well resolved DNA histograms with coefficients of variation below 5%. The estimated nuclear DNA content of the studied ornamental plants is summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA wide range of genome sizes was observed among the species analyzed. The smallest genome size was in \u003cem\u003eAlocasia wentii\u003c/em\u003e (New Guinea Shield), with a DNA content of 0.302 pg (approximately 295 Mbp), whereas \u003cem\u003ePilea cadierei\u003c/em\u003e (Aluminium Plant) exhibited the largest genome size of 2.461 pg (approximately 2407 Mbp). Intermediate genome sizes were observed in \u003cem\u003eClerodendrum chinense\u003c/em\u003e, \u003cem\u003eCodiaeum variegatum\u003c/em\u003e, \u003cem\u003eSpathiphyllum kochii\u003c/em\u003e, and \u003cem\u003eCalathea zebrina\u003c/em\u003e, with DNA contents ranging from approximately 0.676 to 1.964 pg (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The results demonstrate substantial interspecific variation in genome size among commonly cultivated ornamental plants. Such variation in nuclear DNA content is a well-recognized feature of angiosperms and reflects differences in genome organization, repetitive DNA content, and evolutionary history (Sliwinska et al., 2018). Genome size has been linked to influence several biological traits, including cell size, growth rate, and developmental timing, which may indirectly affect ornamental characteristics such as leaf size, plant architecture, and flowering behaviour. In this study, PI staining was employed for genome size estimation due to its stoichiometric binding to double-stranded DNA. In contrast, DAPI may introduce bias associated to base composition particularly differences in GC/AT content (Basak et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nix et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFrom a breeding perspective, nuclear DNA content can support the selection of compatible parental lines, assist in planning hybridization strategies, and support approaches involving polyploidy for trait improvement. The observed genome size diversity among the studied species suggests that the ornamental plants are genetically diverse group with considerable scope for breeding and improvement. Additionally, genome size estimation using flow cytometry is rapid and easy (Nix et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Future studies can integrate flow cytometry with complementary cytogenetic and molecular approaches such as chromosome analysis, ploidy determination, and genome sequencing to gain deeper insights into understand genome structure, organisation and evolutionary relationship among ornamental plants (Zheng et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kishi-Kaboshi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHSK: Analysed, data curation, Manuscript- writing original RK: Analysis, data curationSM: Manuscript- writing original; review editingHK : Supervision, Manuscript- review editing\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThe authors acknowledge the Central Imaging \u0026amp; Flow Cytometry of National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bengaluru.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLeitch, I. J, Johnston E, Pellicer J, Hidalgo O, Bennett MD. 2019. Angiosperm DNA C-values database (release 9.0, Apr 2019) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cvalues.science.kew.org/\u003c/span\u003e\u003cspan address=\"https://cvalues.science.kew.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDoležel, J., Bartoš, J. A. N. Plant DNA flow cytometry and estimation of nuclear genome size. Ann. Bot. 2005, 95(1), 99\u0026ndash;110.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBasak, S., Krishnamurthy, H., Rangan, L., Genome size variation among 3 selected genera of Zingiberoideae. Meta Gene 2018, 15, 42\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNix, J., Ranney, T. G., Lynch, N. P., Chen, H., Flow cytometry for estimating plant genome size: revisiting assumptions, sources of variation, reference standards, and best practices. J. Am. Soc. Hortic. Sci. 2024, 149(3), 131\u0026ndash;141.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan de Peer, Y., Ashman, T. L., Soltis, P. S., Soltis, D. E., Polyploidy: an evolutionary and ecological force in stressful times. Plant Cell, 2021, 33(1), 11\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, J., Ornamental plant research inaugural editorial. Ornamental Plant Research, 2021, 1(1), 1\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDoležel, J., Greilhuber, J., Suda, J., Estimation of nuclear DNA content in plants using flow cytometry. Nat. Protoc. 2007, 2(9), 2233\u0026ndash;2244.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSliwinska, E., Flow cytometry-a modern method for exploring genome size and nuclear DNA synthesis in horticultural and medicinal plant species. Folia Hortic. 2018, 30(1), 103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng, T., Li, P., Li, L., Zhang, Q., Research advances in and prospects of ornamental plant genomics. Hortic. Res. 2021, 8(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKishi-Kaboshi, M., Aida, R., Sasaki, K., Genome engineering in ornamental plants: current status and future prospects. Plant Physiol. Biochem. 2018, 131, 47\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Flow cytometry, Ornamental plants, Genome size, Nuclear DNA content, Plant breeding","lastPublishedDoi":"10.21203/rs.3.rs-9111048/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9111048/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGenome size is a key genomic trait influencing plant growth, development, and breeding potential. Here, nuclear DNA content of six commonly cultivated ornamental plant species was estimated using flow cytometry using propidium iodide staining. Nuclei were isolated from young leaf tissues and stained with propidium iodide (PI). Chicken red blood cells served as an internal reference standard for genome size calculation. All samples produced high-quality DNA histograms with coefficients of variation below 5%. Genome size considerably varied among the studies species, ranging from 0.302 pg in 0.302 pg in \u003cem\u003eAlocasia wentii\u003c/em\u003e to 2.461 pg in \u003cem\u003ePilea cadierei\u003c/em\u003e. Intermediate genome sizes were observed in \u003cem\u003eClerodendrum chinense\u003c/em\u003e, \u003cem\u003eCodiaeum variegatum\u003c/em\u003e, \u003cem\u003eSpathiphyllum kochii\u003c/em\u003e, and \u003cem\u003eCalathea zebrina\u003c/em\u003e. The observed variation suggests genomic diversity in ornamental plants and demonstrates the flow cytometry as a tool, for rapid estimation of genome size.\u003c/p\u003e","manuscriptTitle":"Determining the genome content of ornamental plants using flow cytometry","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-13 08:43:17","doi":"10.21203/rs.3.rs-9111048/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"18b1e09e-8d67-4103-9de9-3f4978ca2862","owner":[],"postedDate":"April 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T08:43:17+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-13 08:43:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9111048","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9111048","identity":"rs-9111048","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}