African Journal of Biotechnology Vol. 5 (18), pp. 1594-1599, 18 September 2006   

ISSN 1684–5315 © 2006 Academic Journals        

Full Length Research Paper

Efficient production of transgenic soybean (Glycine max [L] Merrill) plants mediated via whisker-supersonic (WSS) method

Mutasim M. Khalafalla^1,2*, Hany A. El-Shemy^1,3, Shaikh M. Rahman¹, Masayoshi Teraishi⁴, Hisakazu Hasegawa⁵, Teruhiko Terakawa⁵ and Masao Ishimoto^1,6

¹National Agricultural Research Center for Western Region, 6-12-1 Nishifukatsu, Fukuyama, Hiroshima 721-8514, Japan.

²Commission for Biotechnology and Genetic Engineering, P.O. Box 2404 Khartoum, Sudan.

³Biochemistry Department, Faculty of Agriculture, Cairo University, 12613 Giza, Egypt.

⁴Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.

⁵Hokko chemical industry co., 2165 Toda, Atsugi, Kanagawa 243-0023, Japan.

⁶National Agricultural Research Center for Hokkaido region, 1 Hitsujigaoka, Toyohira-ku, Sappro, Hokkaido 062-8555, Japan.

*Corresponding authors E-mail: mutasim@myway.com

Accepted 28 July, 2006

The present study was designed to evaluate the transformation efficiency and proof the capability of whisker supersonic (WSS) method as an alternative option for soybean (Glycine max [L] Merrill) transformation. We compared soybean transformation efficiency obtained by WSS-mediated with that of particle bombardment transformation by carrying out molecular analysis of the T₀ plants in two independent experiments. For this, we used for both transformation techniques the same genotype, the same plasmid and the same selection method. To assess the efficiency of soybean genetic transformation, we evaluated the efficiency of multi gene transformation by the selection with hygromycin and the expression of green fluorescent protein [sGFP (S65T)] resulted from both techniques. Regenerable embryogenic cells were induced from immature cotyledons of soybean c.v Jack on MSD40 media within 3 weeks then proliferated on FN lite liquid media and engineered with pUHG gene construct through both WSS and particle bombardment-mediated transformation. The pUHG was constructed with pUC 19 and contain the hpt gene conferring resistance to hygromycin as a selective marker and sGFP(S65T) as a reporter gene. Fluorescence microscopy screening after the selection of hygromycin, identified the clearly expression of sGFP(S65T) in the transformed soybean embryos. Stable integration of the transgenes was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. The average transformation efficiency achieved with WSS was higher than that obtained by particle bombardment and hence it may represent an alternative method for soybean transformation.

Key words: Glycine max, whisker supersonic, particle bombardment, transgenic.