Agrobacterium tumefaciens – Mediated transformation of Woodfordia fruticosa (L.) Kurz
In the present study, a protocol for Agrobacterium tumefaciens-mediated transformation has been optimized for Woodfordia fruticosa (L.) Kurz. Precultured axenic leaf segments were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with β-glucuronidase (uidA) co...
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| Format: | Article |
| Language: | English |
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Elsevier
2015-12-01
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| Series: | Journal of Genetic Engineering and Biotechnology |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S1687157X15000347 |
| _version_ | 1827279764601176064 |
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| author | Mallesham Bulle Deepa Rathakatla Raghuvardhan Lakkam Venugopal Rao Kokkirala Mahender Aileni Zhang Peng Sadanandam Abbagani |
| author_facet | Mallesham Bulle Deepa Rathakatla Raghuvardhan Lakkam Venugopal Rao Kokkirala Mahender Aileni Zhang Peng Sadanandam Abbagani |
| author_sort | Mallesham Bulle |
| collection | DOAJ |
| description | In the present study, a protocol for Agrobacterium tumefaciens-mediated transformation has been optimized for Woodfordia fruticosa (L.) Kurz. Precultured axenic leaf segments were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with β-glucuronidase (uidA) containing intron as the reporter gene and hygromycin phosphotransferase (hpt) as a selectable marker gene. After 3 days of co-cultivation, leaf segments were cultured on MS medium containing Thidiazuron (TDZ 4.54 μM) and Indole-3-acetic acid IAA (1.14 μM) + 20 mg/l hygromycin + 200 mg/l cefotaxime (PTSM1) for 4 weeks (includes a single subculture onto the same medium at a 2 week interval). They were subsequently cultured for 3 weeks on MS medium containing Thidiazuron (TDZ 4.54 μM) and Indole-3-acetic acid IAA (1.14 μM) + 25 mg/l hygromycin + 100 mg/l cefotaxime (PTSM2) medium for further development and shoot elongation. The hygromycin resistant shoots were rooted on a rooting medium (PTRM) containing half strength MS medium + 4.90 μM IBA + 25 mg/l hygromycin. A highest transformation efficiency of 44.5% with a mean number of 2.6 transgenic shoots per explant was achieved. Successful transformation was confirmed by the histochemical GUS activity of the regenerated shoots, PCR and RT-PCR analysis using respective primers. Southern blot analysis revealed that the hpt gene integrated into the genome of transgenic W. fruticosa. Establishment of genetic transformation protocol may facilitate the improvement of this medicinal plant in terms of enhancement of secondary metabolites. |
| first_indexed | 2024-04-24T08:24:28Z |
| format | Article |
| id | doaj.art-1df51aaac84b4903bb3262a8455e7bad |
| institution | Directory Open Access Journal |
| issn | 1687-157X |
| language | English |
| last_indexed | 2024-04-24T08:24:28Z |
| publishDate | 2015-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Genetic Engineering and Biotechnology |
| spelling | doaj.art-1df51aaac84b4903bb3262a8455e7bad2024-04-16T22:58:26ZengElsevierJournal of Genetic Engineering and Biotechnology1687-157X2015-12-0113220120710.1016/j.jgeb.2015.09.001Agrobacterium tumefaciens – Mediated transformation of Woodfordia fruticosa (L.) KurzMallesham Bulle0Deepa Rathakatla1Raghuvardhan Lakkam2Venugopal Rao Kokkirala3Mahender Aileni4Zhang Peng5Sadanandam Abbagani6Plant Biotechnology Research Unit, Department of Biotechnology, Kakatiya University, Warangal 506 009, IndiaPlant Biotechnology Research Unit, Department of Biotechnology, Kakatiya University, Warangal 506 009, IndiaPlant Biotechnology Research Unit, Department of Biotechnology, Kakatiya University, Warangal 506 009, IndiaPlant Biotechnology Research Unit, Department of Biotechnology, Kakatiya University, Warangal 506 009, IndiaPlant Biotechnology Research Unit, Department of Biotechnology, Kakatiya University, Warangal 506 009, IndiaNational Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, ChinaPlant Biotechnology Research Unit, Department of Biotechnology, Kakatiya University, Warangal 506 009, IndiaIn the present study, a protocol for Agrobacterium tumefaciens-mediated transformation has been optimized for Woodfordia fruticosa (L.) Kurz. Precultured axenic leaf segments were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with β-glucuronidase (uidA) containing intron as the reporter gene and hygromycin phosphotransferase (hpt) as a selectable marker gene. After 3 days of co-cultivation, leaf segments were cultured on MS medium containing Thidiazuron (TDZ 4.54 μM) and Indole-3-acetic acid IAA (1.14 μM) + 20 mg/l hygromycin + 200 mg/l cefotaxime (PTSM1) for 4 weeks (includes a single subculture onto the same medium at a 2 week interval). They were subsequently cultured for 3 weeks on MS medium containing Thidiazuron (TDZ 4.54 μM) and Indole-3-acetic acid IAA (1.14 μM) + 25 mg/l hygromycin + 100 mg/l cefotaxime (PTSM2) medium for further development and shoot elongation. The hygromycin resistant shoots were rooted on a rooting medium (PTRM) containing half strength MS medium + 4.90 μM IBA + 25 mg/l hygromycin. A highest transformation efficiency of 44.5% with a mean number of 2.6 transgenic shoots per explant was achieved. Successful transformation was confirmed by the histochemical GUS activity of the regenerated shoots, PCR and RT-PCR analysis using respective primers. Southern blot analysis revealed that the hpt gene integrated into the genome of transgenic W. fruticosa. Establishment of genetic transformation protocol may facilitate the improvement of this medicinal plant in terms of enhancement of secondary metabolites.http://www.sciencedirect.com/science/article/pii/S1687157X15000347A. tumefaciensWoodfordia fruticosaLeaf segmentsβ-GlucuronidasehptSouthern blotting |
| spellingShingle | Mallesham Bulle Deepa Rathakatla Raghuvardhan Lakkam Venugopal Rao Kokkirala Mahender Aileni Zhang Peng Sadanandam Abbagani Agrobacterium tumefaciens – Mediated transformation of Woodfordia fruticosa (L.) Kurz Journal of Genetic Engineering and Biotechnology A. tumefaciens Woodfordia fruticosa Leaf segments β-Glucuronidase hpt Southern blotting |
| title | Agrobacterium tumefaciens – Mediated transformation of Woodfordia fruticosa (L.) Kurz |
| title_full | Agrobacterium tumefaciens – Mediated transformation of Woodfordia fruticosa (L.) Kurz |
| title_fullStr | Agrobacterium tumefaciens – Mediated transformation of Woodfordia fruticosa (L.) Kurz |
| title_full_unstemmed | Agrobacterium tumefaciens – Mediated transformation of Woodfordia fruticosa (L.) Kurz |
| title_short | Agrobacterium tumefaciens – Mediated transformation of Woodfordia fruticosa (L.) Kurz |
| title_sort | agrobacterium tumefaciens mediated transformation of woodfordia fruticosa l kurz |
| topic | A. tumefaciens Woodfordia fruticosa Leaf segments β-Glucuronidase hpt Southern blotting |
| url | http://www.sciencedirect.com/science/article/pii/S1687157X15000347 |
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