Aseptic establishment and in vitro germination of Theobroma cacao L.: Redox strategies to overcome recalcitrance

Aseptic establishment and in vitro germination of Theobroma cacao L.: Redox strategies to overcome recalcitrance | 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 Aseptic establishment and in vitro germination of Theobroma cacao L.: Redox strategies to overcome recalcitrance Daniel Martín-Vargas, Mariana Gabriela Andrade, Moraiza Casado-Chacín, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8711263/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 In vitro cultivation of Theobroma cacao L. poses critical challenges, primarily due to the recalcitrant nature of its seeds and the high susceptibility of its tissues to phenolic oxidation. This research evaluated the effect of bioactive modulating agents (cysteine, ascorbic acid, and activated charcoal) and genotypic factors on in vitro germination. By employing an optimized surface disinfection methodology, 100 % asepsis was achieved, significantly surpassing historical records of contamination-related losses (up to 63 %). Germination efficiency reached 86.25 %, being significantly influenced by the Genotype × Environment interaction, where the 'Ocumare 61' cultivar demonstrated superior morphogenic competence. Statistical analysis validated robust mathematical models (R 2 adj of 73.77 % for germination and 81.05 % for oxidation), confirming that supplementation with activated charcoal effectively neutralized the cytotoxic quinone cascade through physical adsorption—a primary limiting factor in the 'Ocumare 60' cultivar. Response surface analysis identified the optimal performance in the M3 treatment with 'Ocumare 61'. We conclude that asepsis control and the stability of the redox microenvironment are decisive conditions for unlocking the germinative potential of cacao seeds. These findings establish a predictive framework that strengthens protocols for mass micropropagation and the conservation of elite germplasm. bioactive modulating agents genotype-environment interaction redox microenvironment recalcitrance phenolic oxidation Full Text 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. 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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-8711263","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":588979550,"identity":"b67d86a1-2f73-4dc3-b6c0-229d62423f41","order_by":0,"name":"Daniel Martín-Vargas","email":"","orcid":"","institution":"Venezuelan Institute for Scientific Research","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Martín-Vargas","suffix":""},{"id":588979551,"identity":"9249ac7b-213f-4552-baaa-87d9bd6ea82a","order_by":1,"name":"Mariana Gabriela Andrade","email":"","orcid":"","institution":"Venezuelan Institute 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phenolic oxidation","lastPublishedDoi":"10.21203/rs.3.rs-8711263/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8711263/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003ecultivation of \u003cem\u003eTheobroma cacao\u003c/em\u003e L. poses critical challenges, primarily due to the recalcitrant nature of its seeds and the high susceptibility of its tissues to phenolic oxidation. This research evaluated the effect of bioactive modulating agents (cysteine, ascorbic acid, and activated charcoal) and genotypic factors on \u003cem\u003ein vitro\u003c/em\u003e germination. By employing an optimized surface disinfection methodology, 100 % asepsis was achieved, significantly surpassing historical records of contamination-related losses (up to 63 %). Germination efficiency reached 86.25 %, being significantly influenced by the Genotype × Environment interaction, where the 'Ocumare 61' cultivar demonstrated superior morphogenic competence. Statistical analysis validated robust mathematical models (R\u003csup\u003e2\u003c/sup\u003e\u0026nbsp;\u003csub\u003eadj\u003c/sub\u003e\u0026nbsp;of 73.77 % for germination and 81.05 % for oxidation), confirming that supplementation with activated charcoal effectively neutralized the cytotoxic quinone cascade through physical adsorption—a primary limiting factor in the 'Ocumare 60' cultivar. 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These findings establish a predictive framework that strengthens protocols for mass micropropagation and the conservation of elite germplasm.\u003c/p\u003e","manuscriptTitle":"Aseptic establishment and in vitro germination of Theobroma cacao L.: Redox strategies to overcome recalcitrance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-11 23:06:30","doi":"10.21203/rs.3.rs-8711263/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":"d0d92504-77f8-4572-b639-8db8f3e1d70f","owner":[],"postedDate":"February 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-17T11:41:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-11 23:06:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8711263","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8711263","identity":"rs-8711263","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}