Optimization of protoplast isolation and transient expression systems for lettuce (Lactuca sativa L.) and other important dicotyledon vegetables

Optimization of protoplast isolation and transient expression systems for lettuce (Lactuca sativa L.) and other important dicotyledon vegetables | 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 Optimization of protoplast isolation and transient expression systems for lettuce (Lactuca sativa L.) and other important dicotyledon vegetables Lei Xiang, Rui-Xin Li, Qing-Jun Zheng, Zan-Tang Huang, Peng-Fei Yu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3993570/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 Plant protoplasts-based platform shows high totipotency, sensitivity, and versatility, and thus promotes botanical advances. However, an efficient protoplasts-based platform remains a challenge for many economically significant dicotyledon vegetables such as lettuce ( Lactuca sativa L.). In the present study, a highly efficient protocol was optimized and developed for isolation of protoplasts from various tissues (leaf, shoot apex, and root tips) of different lettuce subtypes (romaine, loose-leaf, and head lettuces) by optimizing the major factors affecting protoplast yield. It provided the protoplasts yield up to 1.95× 10 7 /g (fw) and viable cells > 85%, which are 3–10 times higher than that of previous report. This protocol was also successfully applied to other dicotyledon plants for efficient isolation of protoplasts from their leaves and shoot apices, including Arabidopsis thaliana , celery cabbage ( brassica pekinensis ), and bok choy ( Brassica parachinensis ). Furthermore, the optimized polyethylene glycol-mediated transient expression system (TES) based on the lettuce shoot apex showed a high transfection efficiency (> 80%), achieving protein subcellular localization of four typical plasma membrane genes and their induced expression function investigations in lettuce. The optimized protoplast isolation protocol and TES provide a versatile technique for functional analyses of genes in lettuces and other important dicotyledon vegetables. Dicotyledon plant Lactuca sativa L. protoplast isolation transient expression systems subcellular localization Full Text Additional Declarations No competing interests reported. Supplementary Files Supplementary.docx 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. 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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-3993570","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":276188485,"identity":"b63afe2a-4b0c-414e-a256-82bc2beffd9f","order_by":0,"name":"Lei Xiang","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Xiang","suffix":""},{"id":276188486,"identity":"26ec6155-66d0-4eea-9ba3-75d5ae0eef34","order_by":1,"name":"Rui-Xin Li","email":"","orcid":"","institution":"Jinan 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vegetables","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"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":"Dicotyledon plant, Lactuca sativa L., protoplast isolation, transient expression systems, subcellular localization","lastPublishedDoi":"10.21203/rs.3.rs-3993570/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3993570/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlant protoplasts-based platform shows high totipotency, sensitivity, and versatility, and thus promotes botanical advances. However, an efficient protoplasts-based platform remains a challenge for many economically significant dicotyledon vegetables such as lettuce (\u003cem\u003eLactuca sativa\u003c/em\u003e L.). In the present study, a highly efficient protocol was optimized and developed for isolation of protoplasts from various tissues (leaf, shoot apex, and root tips) of different lettuce subtypes (romaine, loose-leaf, and head lettuces) by optimizing the major factors affecting protoplast yield. It provided the protoplasts yield up to 1.95\u0026times; 10\u003csup\u003e7\u003c/sup\u003e /g (fw) and viable cells\u0026thinsp;\u0026gt;\u0026thinsp;85%, which are 3\u0026ndash;10 times higher than that of previous report. This protocol was also successfully applied to other dicotyledon plants for efficient isolation of protoplasts from their leaves and shoot apices, including \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, celery cabbage (\u003cem\u003ebrassica pekinensis\u003c/em\u003e), and bok choy (\u003cem\u003eBrassica parachinensis\u003c/em\u003e). Furthermore, the optimized polyethylene glycol-mediated transient expression system (TES) based on the lettuce shoot apex showed a high transfection efficiency (\u0026gt;\u0026thinsp;80%), achieving protein subcellular localization of four typical plasma membrane genes and their induced expression function investigations in lettuce. The optimized protoplast isolation protocol and TES provide a versatile technique for functional analyses of genes in lettuces and other important dicotyledon vegetables.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Optimization of protoplast isolation and transient expression systems for lettuce (Lactuca sativa L.) and other important dicotyledon vegetables","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-08 19:17:46","doi":"10.21203/rs.3.rs-3993570/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":"26230012-6012-4ea1-9164-8986391d1a62","owner":[],"postedDate":"March 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-04T17:45:16+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-08 19:17:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3993570","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3993570","identity":"rs-3993570","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}