African Journal of Biotechnology Vol. 6 (22), pp. 2578-2584, 19 November 2007   

ISSN 1684–5315 © 2007 Academic Journals        

Full Length Research Paper

Optimization of Agrobacterium-mediated transformation parameters for sweet potato embryogenic callus using β-glucuronidase (GUS) as a reporter

Yujun Xing^1,2, Qing Yang¹, Qin Ji²*, Yuming Luo², Yunfeng Zhang², Ke Gu² and Dengzhan Wang²

¹College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China.

²Department of Biology, Huaiyin Teachers College, Huaiyin, 223300, China.

*Corresponding author. E-mail: jiqin1003@yahoo.com.cn. Tel: +86-517-3525382. Fax: +86-517-3525992.

Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; AS, acetosyringone; CIM, callus induced medium; GUS, β-Glucuronidase; MS, Murashige and Skoog; rpm, rotate per minute; CCM, co-cultured medium.

Accepted 23 October, 2007

Agrobacterium-mediated transformation factors for sweet potato embryogenic calli were optimized using β-glucuronidase (GUS) as a reporter. The binary vector pTCK303 harboring the modified GUS gene driven by the CaMV 35S promoter was used. Transformation parameters were optimized including bacterial concen-tration, pre-culture period, co-cultivation period, immersion time, acetosyringone (AS) concentration and mannitol treated time. Results were obtained based on the percentage of GUS expression. Agrobacterium tumefaciens strain EHA105 at concentration OD_{600 nm} = 0.8 showed the highest virulence on sweet potato embryogenic callus. Four days of pre-culture, four days of co-cultivation, 10 min of immersion, 200 μM acetosyringone and 60 min of mannitol-treated embryogenic callus gave the highest percentage of GUS positive transformants.

Key words: Agrobacterium-mediated, transformation parameters, sweet potato embryogenic callus, β-glucuronidase.