Global Advanced Research Journal of Agricultural Science (GARJAS) ISSN: 2315-5094
November 2015 Vol. 4(11): pp. 761-768
Copyright © 2015 Global Advanced Research Journals
Full Length Research Paper
Enhanced efficiency of Agrobacterium-mediated transformation by sulfamethazine treatment in ravenna grass, Erianthus ravennae (L.) Beauv.
Kazuki Shimomae1), So Makabe1), Tanaphol Boriboonkaset1), Dong Poh Chin1),Tomoko Igawa1), Raham Sher Khan2), Masahiro Mii1), 3) and Ikuo Nakamura1)
1) Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba 271-8510, Japan.
2) Department of Biotechnology, Abdul Wali Khan University, Mardan 23200, Pakistan
3) Center for Environment, Health and Field Sciences, Chiba University, 6-2-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.
Corresponding Author's Email:email@example.com;Tel & Fax: +81-47-308-8852
Accepted 06 November, 2015
Agrobacterium-mediated transformation system was developed in ravenna grass [Erianthus ravennae (L.) Beauv.], a potential source of bioenergy. Agrobacterium tumefaciens EHA101 harboring pGWB-UbiGFP, which contains the maize ubiquitin promoter (Ubi)-driven green fluorescent protein (GFP) gene and cauliflower mosaic virus (CaMV) 35S promoter-driven hygromycin phosphotransferase (hpt) gene, was used for transformation. Calli cultured in liquid medium were infected and co-cultivated for 3 days on medium with all MS-salts removed. The transformed calli were selected on MS media containing 50 mg l-1 hygromycin. After 6 months of the selection, 43 hygromycin-resistant callus lines (8.5%) were obtained from 500 infected calli. The GFP florescence was weak in the all hygromycin-resitant calli, likely due to the silencing of GFP transgene. These calli were treated with 30 µ M sulfamethazine (SMZ) for 2 months, resulting that SMZ-treated calli exhibited stronger GFP florescence than non-treated calli. Finally, seven shoots (16.3%) were regenerated from the SMZ-treated callus lines, while only one shoot (2.3%) was obtained from the non-treated callus lines. GFP florescence was also observed in leaves, roots, and shoot apex of plants regenerated from the SMZ-treated calli. Integrations of GFP and hpt transgenes into the genome of regenerated plants were confirmed by PCR and Sothern blot analyses. The expressions of these transgenes were confirmed by RT-PCR.
Keywords: Agrobacterium tumefaciens, Erianthus, transformation, sulfamethazine, MS-free medium.
- Kazuki Shimomae on Google Scholar
- Kazuki Shimomae on Pubmed
- So Makabe on Google Scholar
- So Makabe on Pubmed
- Tanaphol Boriboonkaset on Google Scholar
- Tanaphol Boriboonkaset on Pubmed
- Dong Poh Chin on Google Scholar
- Dong Poh Chin on Pubmed
- Tomoko Igawa on Google Scholar
- Tomoko Igawa on Pubmed
- Raham Sher Khan on Google Scholar
- Raham Sher Khan on Pubmed
- Masahiro Mii on Google Scholar
- Masahiro Mii on Pubmed
- Ikuo Nakamura on Google Scholar
- Ikuo Nakamura on Pubmed