Original Research Production of Haploids and Doubled Haploids from Unfertilized Ovule Culture of Various Wild Species of Gentians (Gentiana spp.)

Original Research Production of Haploids and Doubled Haploids from Unfertilized Ovule Culture of Various Wild Species of Gentians (Gentiana spp.) | 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 Original Article Original Research Production of Haploids and Doubled Haploids from Unfertilized Ovule Culture of Various Wild Species of Gentians (Gentiana spp.) Yutaro Takamura, Ryo Takahashi, Takashi Hikage, Katsunori Hatakeyama, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-181197/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract The production of haploids and doubled haploids (DHs) on unfertilized ovule culture was examined in 19 wild species of gentians ( Gentiana ssp.) classified into eight sections. Of the 19 species including 35 strains, embryo-like structures (ELSs) were obtained in 15 species, and regenerated plants were produced in 11 species. ELS production has varied greatly among the15 species, i.e., 0.5%-79.2% frequency of responding flower buds and 0.01-1.99 ELSs per flower bud. Of the ELS-producing species, almost all were classified into the sects. Pneumonanthe or Cruciata . Species in sect. Pneumonanthe showed higher responses than those in sect. Cruciata . In examining the effect of flower bud stage on ELS production, more than twice as many ELSs were observed at the anther-dehiscent stage than that at the anther-indehiscent stage. Ploidy level was determined in 117 randomly selected regenerated plantlets, which suggests that most were haploid (32.5%) and diploid (46.2%). When 12 diploid plants were examined using simple sequence repeat (SSR) markers, 8 (66.7%) were DH. This study revealed that unfertilized ovule culture can be applied not only on cultivated gentian species but also on a number of wild species. The production of haploids and DHs in wild gentians provides novel prospects for ornamental and/or medicinal gentian breeding. Plant Physiology and Morphology Plant Molecular Biology and Genetics Gentiana Wild species Unfertilized ovule culture Bud stage Doubled haploid Molecular marker Figures Figure 1 Figure 2 Figure 3 Full Text Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the latest manuscript can be downloaded and accessed as a PDF. Tables Due to technical limitations, table 1, 3 and 4 is only available as a download in the Supplemental Files section.</p Table 2 not available with this version Supplementary Files Table1.pdf Table4.pdf Table3.pdf Supple.Table1.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 02 Feb, 2021 Reviewers invited by journal 31 Jan, 2021 First submitted to journal 28 Jan, 2021 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-181197","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Original Article","associatedPublications":[],"authors":[{"id":9899203,"identity":"87d49add-1015-4c7f-81fa-01d354b487ae","order_by":0,"name":"Yutaro Takamura","email":"","orcid":"","institution":"Hachimantai City Floricultural Research and Development Center","correspondingAuthor":false,"prefix":"","firstName":"Yutaro","middleName":"","lastName":"Takamura","suffix":""},{"id":9899204,"identity":"a2672b30-055c-4e8d-bd2a-10b7d63eeb99","order_by":1,"name":"Ryo Takahashi","email":"","orcid":"","institution":"Hachimantai City Floricultural Research and Development Center","correspondingAuthor":false,"prefix":"","firstName":"Ryo","middleName":"","lastName":"Takahashi","suffix":""},{"id":9899205,"identity":"883e2b66-6d69-4472-ae32-d38ef3f08ded","order_by":2,"name":"Takashi Hikage","email":"","orcid":"","institution":"Haichimantai City Floricultural Research and Development Center","correspondingAuthor":false,"prefix":"","firstName":"Takashi","middleName":"","lastName":"Hikage","suffix":""},{"id":9899206,"identity":"a8106277-6ba2-4b81-91b6-259a21a7abb7","order_by":3,"name":"Katsunori Hatakeyama","email":"","orcid":"","institution":"Iwate University: Iwate Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Katsunori","middleName":"","lastName":"Hatakeyama","suffix":""},{"id":9899207,"identity":"d94c2303-052c-47cc-89b9-cc1de3cbe46d","order_by":4,"name":"Yoshihito Takahata","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIiWNgGAWjYHACZgbGBiDF3tiAJMiDRwMbRIsEA89BoJYEkrRIgJQn4FEJAwb3mx8b/NxhV8c/83GbxM8fNgwGB5gffmCQuYNbyzE248TeM8kSErcT2yR7EtKAWtiMgc58hkcLg/EB3jZmCQaglhs8CYfrNxxgMAP65TAeLeyfD/5tq5eQv3mw7eafhP9AW9i/EdDCY5zM23ZYwuAGY9ttnoQDQC08+G2RPJZTbCzbdlxy45nE9t8yackMkod5iiUS8PiF7/DxzZJv26r55Y4ff2z4xsaOge94+8YPH3twh5jCAQwhZiBO7MEUhwH5BuziP3BrGQWjYBSMghEHAGWIV5vP4dbmAAAAAElFTkSuQmCC","orcid":"","institution":"Iwate University","correspondingAuthor":true,"prefix":"","firstName":"Yoshihito","middleName":"","lastName":"Takahata","suffix":""}],"badges":[],"createdAt":"2021-01-29 02:20:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-181197/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-181197/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":5837199,"identity":"1cc3d342-3274-4091-837d-2adc4f858e4f","added_by":"auto","created_at":"2021-02-10 19:21:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10281259,"visible":true,"origin":"","legend":"Embryo like structure (ELS) induction and plant regeneration from unfertilized ovule culture of wild gentian (G. septemfida). a Gentian flower at the time of harvest. b Two stages of flower buds; anther-indehiscent stage (left) and anther-dehiscent stage (right). c Ovules cultured on 1/2 NLN solid medium. d A ELS emerged from ovules; e A plantlet regenerated from ELS. f A regenerated plant. g, h Flow cytometoric analysis; g showing a haploid, h showing a diploid. Bars in a = 50 mm, b, c, e = 10 mm, d = 1 mm, f = 30 mm. ","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-181197/v1/215d6299ddf7c525ca616660.png"},{"id":5836869,"identity":"419fb631-791a-482f-9b19-87b61821b6df","added_by":"auto","created_at":"2021-02-10 19:15:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":" Effect of flower bud stage (anther-indehiscence vs. anther-dehiscence) on ELS production and plant regeneration in unfertilized ovule culture of wild gentians over two years (2017 and 2018) examinations. a, c No. of ELSs per flower bud. b, d No. of regenerated plants per flower bud. a, b 2017 and c, d 2018. Error bars present the standard error (SE). Asterisks indicate statistically significant differences at the 0.05 level according to Student’s t-test. ","description":"","filename":"placeholderimage.png","url":"https://assets-eu.researchsquare.com/files/rs-181197/v1/80ab27e0374eefcf0f3f03d9.png"},{"id":5837084,"identity":"be9f66a9-1268-40eb-a699-50e7ec7dfe39","added_by":"auto","created_at":"2021-02-10 19:18:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1644321,"visible":true,"origin":"","legend":"Analysis of genetic constitution of diploid plants derived from unfertilized ovule culture in G. septemfida. a G. septemfida ‘7-131-1’ analyzed using Gtm12 SSR marker. b G. septemfida ‘7-131-2’ nalyzed using Gtm68 SSR marker. M: 100bp molecular marker. ","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-181197/v1/bf79b40498f75c15089ba512.png"},{"id":13583046,"identity":"2b62b7b6-b6fd-4cca-a1ce-b8ebd32c4003","added_by":"auto","created_at":"2021-09-17 04:33:33","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":404621,"visible":true,"origin":"","legend":"","description":"","filename":"Unfertilizedovulecultureofwildgentians.pdf","url":"https://assets-eu.researchsquare.com/files/rs-181197/v1_covered.pdf"},{"id":5837496,"identity":"86cc4c8f-7d10-4d38-9df7-02a90c7cfb49","added_by":"auto","created_at":"2021-02-10 19:24:44","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":231666,"visible":true,"origin":"","legend":"","description":"","filename":"Unfertilizedovulecultureofwildgentians.pdf","url":"https://assets-eu.researchsquare.com/files/rs-181197/v1_stamped.pdf"},{"id":5837198,"identity":"5c2cc81e-d2a6-4be1-b4ab-94f3e65ae414","added_by":"auto","created_at":"2021-02-10 19:21:39","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":71535,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-181197/v1/24b2250ab8b679fc8c8e356d.pdf"},{"id":5837079,"identity":"579f247e-4632-42ea-87c2-b67bd12b1569","added_by":"auto","created_at":"2021-02-10 19:18:39","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":62954,"visible":true,"origin":"","legend":"","description":"","filename":"Table4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-181197/v1/3146e37d25091f530548fd3d.pdf"},{"id":5837080,"identity":"bc35735c-ce15-4c2c-9c51-6a67c0c4694a","added_by":"auto","created_at":"2021-02-10 19:18:39","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":74474,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-181197/v1/f606790339b0d458c3e1b3a3.pdf"},{"id":5837082,"identity":"c1b6d8e3-061c-4e95-b57f-b2d7116b15a7","added_by":"auto","created_at":"2021-02-10 19:18:39","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":53123,"visible":true,"origin":"","legend":"","description":"","filename":"Supple.Table1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-181197/v1/51701318296e039fbcaa3b9c.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eOriginal Research Production of Haploids and Doubled Haploids from Unfertilized Ovule Culture of Various Wild Species of Gentians (Gentiana\u0026nbsp;spp.)\u003c/p\u003e","fulltext":[{"header":"Full Text","content":"Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the latest manuscript can be downloaded and \u003ca href='/article/rs-181197/latest.pdf' target='_blank'\u003e accessed as a PDF.\u003c/a\u003e"},{"header":"Tables","content":"\u003cp\u003eDue to technical limitations, table 1, 3 and 4 is only available as a download in the Supplemental Files section.\u003c/p\n\u003cp\u003eTable 2 not available with this version\u003cp\u003e "}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"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":"Gentiana, Wild species, Unfertilized ovule culture, Bud stage, Doubled haploid, Molecular marker ","lastPublishedDoi":"10.21203/rs.3.rs-181197/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-181197/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The production of haploids and doubled haploids (DHs) on unfertilized ovule culture was examined in 19 wild species of gentians ( Gentiana ssp.) classified into eight sections. Of the 19 species including 35 strains, embryo-like structures (ELSs) were obtained in 15 species, and regenerated plants were produced in 11 species. ELS production has varied greatly among the15 species, i.e., 0.5%-79.2% frequency of responding flower buds and 0.01-1.99 ELSs per flower bud. Of the ELS-producing species, almost all were classified into the sects. Pneumonanthe or Cruciata . Species in sect. Pneumonanthe showed higher responses than those in sect. Cruciata . In examining the effect of flower bud stage on ELS production, more than twice as many ELSs were observed at the anther-dehiscent stage than that at the anther-indehiscent stage. Ploidy level was determined in 117 randomly selected regenerated plantlets, which suggests that most were haploid (32.5%) and diploid (46.2%). When 12 diploid plants were examined using simple sequence repeat (SSR) markers, 8 (66.7%) were DH. This study revealed that unfertilized ovule culture can be applied not only on cultivated gentian species but also on a number of wild species. The production of haploids and DHs in wild gentians provides novel prospects for ornamental and/or medicinal gentian breeding.","manuscriptTitle":"Original Research Production of Haploids and Doubled Haploids from Unfertilized Ovule Culture of Various Wild Species of Gentians (Gentiana\u0026nbsp;spp.)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-10 19:15:38","doi":"10.21203/rs.3.rs-181197/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-02-03T00:00:00+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-02-01T00:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2021-01-28T21:20:47+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":"b5dfd63b-1dcc-45e1-9973-92eadfdbf856","owner":[],"postedDate":"February 10th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":2339648,"name":"Plant Physiology and Morphology"},{"id":2339649,"name":"Plant Molecular Biology and Genetics"}],"tags":[],"updatedAt":"2021-04-13T14:17:10+00:00","versionOfRecord":[],"versionCreatedAt":"2021-02-10 19:15:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-181197","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-181197","identity":"rs-181197","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}