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Agrobacterium tumefaciens-mediated genetic transformation of Digitalis purpurea L.

Tijdschriftbijdrage - Tijdschriftartikel

Genetic transformation is a tool of special interest to develop new biotechnological strategies for the production of bio-active compounds such as cardenolides, exclusively obtained from plants. To date, Digitalis plants are the main economically viable source of cardenolides for pharmaceutical industry. This study describes the development of efficient plant regeneration and Agrobacterium-mediated genetic transformation protocols for Digitalis purpurea L. First, a plant regeneration procedure starting from leaf segments of in vitro cultivated plants was established and the minimal inhibitory concentration of G-418 (geneticin) for callus induction was determined. Both leaf segments and callus tissue resulted sensitive to G-418 70 mg.l-1. Afterwards, two Agrobacterium strains were used to test their T-DNA transfer ability on D. purpurea leaf tissues, EHA105 and C58C1RifR (pMP90), both harboring the binary vector pTJK136. Strain C58C1RifR (pMP90) yielded a higher number of transformed plants than EHA105. Successful transformation was confirmed by histochemical beta-glucuronidase (GUS) assays of the putative transgenic tissues and PCR analyses using beta-glucuronidase (uidA)- and neomycin phosphotransferase II (nptII)- specific primers. Southern blot hybridization confirmed the stable integration of nptII gene in the transgenic plants. In total, 518 independent transgenic lines were regenerated with an average of 6.91 transgenic lines per initial leaf segment using C58C1RifR (pMP90) strain. To date, only a few studies have been published on the genetic transformation of Digitalis species. The protocols for plant regeneration and genetic transformation described in this paper will contribute to functional studies for a better understanding of cardenolide biosynthetic pathways and the metabolic engineering of cardenolides to develop high yielding improved genotypes.
Tijdschrift: Plant Biotechnol Rep
ISSN: 1863-5466
Volume: 8
Pagina's: 387-397
Jaar van publicatie:2014
Trefwoorden:foxglove, plant regeneration, Agrobacterium, transgenic plants, cardenolides