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Agrobacterium tumefaciens-mediated genetic transformation of Digitalis purpurea L.
Naivy Pe´rez-Alonso,Elio Jime´nez,Borys Chong-Pe´rez,Alina Capote,Anabel Pe´rez,Yovanny Izquierdo,Geert Angenon 한국식물생명공학회 2014 Plant biotechnology reports Vol.8 No.5
Genetic transformation is a tool of specialinterest for developing new biotechnological strategies forthe production of bio-active compounds such as cardenolides,which are exclusively obtained from plants. To date,Digitalis plants are the main economically viable source ofcardenolides for the pharmaceutical industry. This studydescribes the development of efficient plant regenerationand Agrobacterium-mediated genetic transformation protocolsfor Digitalis purpurea L. First, a plant regenerationprocedure starting from leaf segments of in vitro-cultivatedplants was established and the minimal inhibitory concentrationof G-418 (geneticin) for callus induction wasdetermined. Both leaf segments and callus tissue weresensitive to G-418 70 mg l-1. Afterwards, two Agrobacteriumstrains were used to test their T-DNA transferability on D. purpurea leaf tissues, EHA105 andC58C1RifR (pMP90), both harboring the binary vectorpTJK136. Strain C58C1RifR (pMP90) yielded a highernumber of transformed plants than EHA105. Successfultransformation was confirmed by histochemical b-glucuronidase(GUS) assays of the putative transgenic tissuesand PCR analyses using b-glucuronidase (uidA)- andneomycin phosphotransferase II (nptII)-specific primers. Southern blot hybridization confirmed the stable integrationof the nptII gene in the transgenic plants. In total, 518independent transgenic lines were regenerated with anaverage of 6.91 transgenic lines per initial leaf segmentinfected with A. tumefaciens strain C58C1RifR (pMP90). To date, only a few studies have been published on thegenetic transformation of Digitalis species. The protocolsfor plant regeneration and genetic transformation describedin this paper will contribute to functional studies for abetter understanding of cardenolide biosynthetic pathwaysand the metabolic engineering of cardenolides to develophigh-yielding improved genotypes.
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