Full Length Research Paper

Effects of basal media, salt concentrations, antioxidant supplements and co-effects on the Agrobacterium-mediated transformation efficiency in maize

Hewei Du^{1 2}, Huixia Wu³, Jianbing Yan¹ and Jiansheng Li ¹*

¹China Agricultural University, National Maize Improvement Center of China, Beijing 100193, P.R.China.

²The College of life science, Yangtze University, Jingzhou, Hubei 434025, P.R.China.

³International Maize and Wheat Improvement Center (CIMMYT), Apdo. Postal 6-641, 06600 México, D.F., México.

*Corresponding author. E-mail: lijiansheng@cau.edu.cn. Tel: 86-10-62732442. Fax: 86-10-62733808.

Abbreviations: N6, Chu medium; MS, Murashige and Skoog medium; LS, Linsmaier and Skoog medium; D, Ducan medium; 2,4-D, 2,4-Dichlorophenoxyacetic acid; MES, 2-(N-Morpholino) ethanesulfonic acid; DTT, Dithiothreitol; NAA, α-Naphthalene acetic acid; 6-BA, N6-benzylaminopurine; CTAB, Cetyltrimethyl ammonium bromide; DIG, Digoxin; PVP, Polypyrrolidone; GUS, β-Gluceronidase gene; Bar, Bialaphos resistance gene.

Accepted 15 January, 2010

   Abstract

Transformation efficiency enhancement in maize Agrobacterium-mediated transformation was tested using four different basal media, five levels of N6 salts, two antioxidants and copper sulfate. In the absence of the antioxidants L-cysteine and dithiothreitol (DTT), the frequencies of transient GUS expression was higher using Linsmaier and Skoog (LS) and Murashige and Skoog (MS) media as an alternative to Chu (N6) and Ducan (D). N6 basal medium exhibited better performance in the presence of antioxidants than MS, LS and D basal media. Five different levels of N6 medium salts (10, 30, 50, 70 and 100%) were tested, and the highest transformation efficiency was 15.9% under a 50% salt concentration, followed by 6.4% transformation efficiency with 70 and 3.2% under 100% salt conditions. More than 95% of infected immature embryos exhibited GUS staining under 10 and 30% salt concentrations, however none of the embryos developed into embryogenic callus, indicating that low salt levels favored T-DNA delivery, but not stable transformation. Additions of DTT or L-cysteine, or a combination of L-cysteine and DTT, showed a significant improvement in the frequency of transient GUS expression, however increases were not observed with independent CuSO₄ treatments. Polymerase chain reaction (PCR) and Southern-blot analysis confirmed T-DNA integration into the maize genome.

Key words: Maize (Zea mays L.), Agrobacterium tumefaciens, transformation.