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Protoplast isolation, transient transformation, and flow-cytometric analysis of reporter-gene activation in Cannabis sativa L.

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dc.contributor.authorBeard, Keely Marieen
dc.contributor.authorBoling, Audrey W. H.en
dc.contributor.authorBargmann, Bastiaan O. R.en
dc.date.accessioned2021-04-09T19:13:56Zen
dc.date.available2021-04-09T19:13:56Zen
dc.date.issued2021-06-01en
dc.date.updated2021-04-09T16:02:38Zen
dc.description.abstractCannabis sativa L. is a valuable, up-and-coming industrial crop with a substantially growing market. However, due to an extended period of legal restriction, research with cannabis has been limited, particularly in laboratory settings. Expanding the application of biotechnological techniques to cannabis can facilitate addressing species-specific impediments to improving crop traits and further fundamental understanding of its intricacies. Here, we describe application of protoplast transformation for the study of transient gene expression in a low-THC cannabis cultivar. To produce explant tissue as a source of protoplasts, a method for hormone-free in vitro micropropagation is established. Protoplasts are isolated from young leaves of the micropropagated stocks and transiently transformed with plasmid DNA carrying a fluorescent marker gene. This is the first report of protoplast transformation in this species. A protoplast isolation yield is achieved of up to 2 × 10 cells per gram of leaf material, vitality staining shows that up to 82 % of isolated protoplasts are viable, and quantification of the cells expressing a fluorescent protein indicates that up to 31 % of the cells can be successfully transformed. Additionally, protoplasts are transformed with an auxin-responsive reporter gene and the reaction to treatment with indole-3-acetic acid is quantified using flow cytometry. This work demonstrates that relatively minor modification of standard techniques can be used to study this important emerging crop. 6en
dc.description.versionAccepted versionen
dc.format.extentPages 113360-113360en
dc.format.mimetypeapplication/pdfen
dc.identifier113360 (Article number)en
dc.identifier.doihttps://doi.org/10.1016/j.indcrop.2021.113360en
dc.identifier.issn0926-6690en
dc.identifier.orcidBargmann, Bastiaan [0000-0001-8406-5036]en
dc.identifier.urihttp://hdl.handle.net/10919/102992en
dc.identifier.volume164en
dc.language.isoenen
dc.publisherElsevieren
dc.subjectBiotechnologyen
dc.subject0703 Crop and Pasture Productionen
dc.titleProtoplast isolation, transient transformation, and flow-cytometric analysis of reporter-gene activation in Cannabis sativa L.en
dc.title.serialIndustrial Crops and Productsen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten
dc.type.otherJournal Articleen
dcterms.dateAccepted2021-02-18en
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciencesen
pubs.organisational-group/Virginia Tech/All T&R Facultyen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciences/CALS T&R Facultyen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciences/School of Plant and Environmental Sciencesen
pubs.organisational-group/Virginia Techen

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