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:inakamur@faculty.chiba-u.ac.jp;Tel & Fax: +81-47-308-8852

Accepted 06 November, 2015

 

Abstract

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. 


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