Appl Biochem Biotechnol (2014) 172:1807–1817 DOI 10.1007/s12010-013-0648-6
A Stable and Efficient Agrobacterium tumefaciens-Mediated Genetic Transformation of the Medicinal Plant Digitalis purpurea L. Ying Li & Zhenrui Gao & Chunlan Piao & Kaiwen Lu & Zhiping Wang & Min-Long Cui
Received: 3 September 2013 / Accepted: 6 November 2013 / Published online: 24 November 2013 # Springer Science+Business Media New York 2013
Abstract In this study, we developed a rapid and efficient method for in vitro propagation and Agrobacterium tumefaciens-mediated transformation of Digitalis purpurea L. (syn. foxglove), an important medicinal plant. Mature leaf explants of D. purpurea were used for 100 % adventitious shoot regeneration on Murashige and Skoog (MS) medium supplemented with 1 mg L−1 thidiazuron (TDZ) (a cytokine) and 0.1 mg L−1 1-naphthaleneacetic acid (NAA) (an auxin). Transformation was achieved by inoculating leaf explants with the A. tumefaciens strains GV2260/pBI121 or GV3101/pBI121. The binary vector pBI121 contained the reporter β-glucuronidase gene (GUS) and kanamycin selection marker nptII. Kanamycin-resistant shoots were regenerated directly on the selection medium 4–6 weeks after co-cultivation. Approximately, 52.2 and 60 % of kanamycin-resistant shoots transformed with Agrobacterium strains GV2260 and GV3101, respectively, showed strong GUS staining by histochemical assay. Furthermore, PCR and Southern blot analysis confirmed the presence of nptII and GUS on the chromosome of the transformed D. purpurea plants, and stable GUS expression was detected in the transformants by RT-PCR analysis. This efficient method of shoot regeneration and genetic transformation of D. purpurea will provide a powerful tool to increase and produce valuable components such as digitoxin, digoxin, and digoxigenin in D. purpurea through improved secondary metabolic pathways via a biotechnological approach. Keywords Digitalis purpurea L . Common medicinal plant . Mature leaf explants . High-efficiency shoot regeneration . Agrobacterium tumefaciens-mediated transformation . Stable GUS expression
Y. Li : Z. Gao : C. Piao : M.
A Stable and Efficient Agrobacterium tumefaciens-Mediated Genetic Transformation of the Medicinal Plant Digitalis purpurea L. Ying Li & Zhenrui Gao & Chunlan Piao & Kaiwen Lu & Zhiping Wang & Min-Long Cui
Received: 3 September 2013 / Accepted: 6 November 2013 / Published online: 24 November 2013 # Springer Science+Business Media New York 2013
Abstract In this study, we developed a rapid and efficient method for in vitro propagation and Agrobacterium tumefaciens-mediated transformation of Digitalis purpurea L. (syn. foxglove), an important medicinal plant. Mature leaf explants of D. purpurea were used for 100 % adventitious shoot regeneration on Murashige and Skoog (MS) medium supplemented with 1 mg L−1 thidiazuron (TDZ) (a cytokine) and 0.1 mg L−1 1-naphthaleneacetic acid (NAA) (an auxin). Transformation was achieved by inoculating leaf explants with the A. tumefaciens strains GV2260/pBI121 or GV3101/pBI121. The binary vector pBI121 contained the reporter β-glucuronidase gene (GUS) and kanamycin selection marker nptII. Kanamycin-resistant shoots were regenerated directly on the selection medium 4–6 weeks after co-cultivation. Approximately, 52.2 and 60 % of kanamycin-resistant shoots transformed with Agrobacterium strains GV2260 and GV3101, respectively, showed strong GUS staining by histochemical assay. Furthermore, PCR and Southern blot analysis confirmed the presence of nptII and GUS on the chromosome of the transformed D. purpurea plants, and stable GUS expression was detected in the transformants by RT-PCR analysis. This efficient method of shoot regeneration and genetic transformation of D. purpurea will provide a powerful tool to increase and produce valuable components such as digitoxin, digoxin, and digoxigenin in D. purpurea through improved secondary metabolic pathways via a biotechnological approach. Keywords Digitalis purpurea L . Common medicinal plant . Mature leaf explants . High-efficiency shoot regeneration . Agrobacterium tumefaciens-mediated transformation . Stable GUS expression
Y. Li : Z. Gao : C. Piao : M.
