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

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.