Improvement of Agrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binary vectors

Abstract

High-frequency transformation of maize (Zea mays L.) using standard binary vectors is advantageous for functional genomics and other genetic engineering studies. Recent advances in Agrobacterium tumefaciens-mediated transformation of maize have made it possible for the public to transform maize using standard binary vectors without a need of the superbinary vector. While maize Hi-II has been a preferred maize genotype to use in various maize transformation efforts, there is still potential and need in further improving its transformation frequency. Here we report the enhanced Agrobacterium-mediated transformation of immature zygotic embryos of maize Hi-II using standard binary vectors. This improved transformation process employs low-salt media in combined use with antioxidant L-cysteine alone or L-cysteine and dithiothreitol (DTT) during the Agrobacterium infection stage. Three levels of N6 medium salts, 10, 50, and 100%, were tested. Both 10 and 50% salts were found to enhance the T-DNA transfer in Hi-II. Addition of DTT to the cocultivation medium also improves the T-DNA transformation. About 12% overall and the highest average of 18% transformation frequencies were achieved from a large number of experiments using immature embryos grown in various seasons. The enhanced transformation protocol established here will be advantageous for maize genetic engineering studies including transformation-based functional genomics.

Agrobacterium-mediated genetic transformation of a tropical elite maize line

“…Usually, conditions which allow a high level of expression of this reporter gene are associated with a high rate of production of transgenic events (Hiei at al. 1994, Petrillo et al 2008, Vega et al 2008). …”

“…Although the transformation improvement mechanism is not clear, the use of low salt contents through the infection and cocultivation stages has been shown to improve the transfer of T-DNA (Fry et al 1987, Cheng et al 1997, Armstrong and Rout 2001, Zhang et al 2002, Paz et al 2004, Vega et al 2008). …”

“…The N6 (Chu et al 1975) and MS (Murashige and Skoog 1962) salts are widely used for in vitro cultivation and genetic transformation of maize (Ishida et al 1996, Frame et al 2006, Vega et al 2008, Li et al 2015. Embryogenic calli are normally formed when immature zygotic maize embryos are cultured in these basal media supplemented with auxins, such as 2,4-D or Dicamba (Green and Phillips 1975, Armstrong and Green 1985, Frame et al 2006, Petrillo et al 2008).…”

“…To date, immature zygotic embryos are the explant with the highest regeneration efficiency, and competence for infection by A. tumefaciens and generation of transgenic plants (Frame et al 2006, Vega et al 2008). …”

“…Genetic transformations of maize were performed using culture media 1 and 2 according to Frame et al (2006) and Vega et al (2008), respectively, with few modifications. All culture media are described in Table 1.…”