The development of herbicide-resistant maize: stable Agrobacterium-mediated transformation of maize using explants of type II embryogenic calli

Abstract

One of the limitations to conducting maize Agrobacterium-mediated transformation using explants of immature zygotic embryos routinely is the availability of the explants. To produce immature embryos routinely and continuously requires a well-equipped greenhouse and laborious artificial pollination. To overcome this limitation, an Agrobacterium-mediated transformation system using explants of type II embryogenic calli was developed. Once the type II embryogenic calli are produced, they can be subcultured and/or proliferated conveniently. The objectives of this study were to demonstrate a stable Agrobacterium-mediated transformation of maize using explants of type II embryonic calli and to evaluate the efficiency of the protocol in order to develop herbicide-resistant maize. The type II embryogenic calli were inoculated with Agrobacterium tumefaciens strain C58C1 carrying binary vector pTF102, and then were subsequently cultured on the following media: co-cultivation medium for 1 day, delay medium for 7 days, selection medium for 4 9 14 days, regeneration medium, and finally on germination medium.The T-DNA of the vector carried two cassettes (Ubi promoter-EPSPs ORF-nos and 35S promoter-bar ORFnos). The EPSPs conferred resistance to glyphosate and bar conferred resistance to phosphinothricin. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to phosphinothricin indicated by the growth of putative transgenic calli on selection medium amended with 4 mg l -1 phosphinothricin, northern blot analysis of bar gene, and leaf painting assay for detection of bar gene-based herbicide resistance. Northern blot analysis and leaf painting assay confirmed the expression of bar transgenes in the R 1 generation. The average transformation efficiency was 0.60%. Based on northern blot analysis and leaf painting assay, line 31 was selected as an elite line of maize resistant to herbicide.

Expression of Dengue virus EIII domain-coding gene in maize as an edible vaccine candidate

“…Since a stable and efficient of transformation using immature embryos (Cho et al 2005) or type II callus (Kim et al 2009) of maize had been reported in our V663-33-4128, V663-33-4098, V663-33-4094, V663-33-4034, V663-33-3619, V663-33-3783, V663-33-3326, V663-32-3559, V662-29-3942, V662-33-2784, V662-29-3864 previous study, and transgenic maize expressing EPSP herbicide resistant gene (Cho et al 2007) and Actinobacillus pleuropneumoniae ApxIIA gene as vaccine candidate were developed. By the stable and efficient protocols, putative transgenic plants based on paromomycinresistance were produced, although the transformation frequency was lower than 5.5% (Frame et al 2002) and higher than our previous study with 0.6% (Cho et al 2005;Kim et al 2009). In general, the factors influencing transformation efficiency include plant species, explants types, maturity, researcher skill, selectable marker, etc.…”

Development of Transgenic Maize using Immature Embryos of HiII Genotype as a Vaccine Candidate

“…After that, Mason et al (1996) have been also reported the expression of Norwalk virus capsid protein in tobacco plants and potato tubers. Specially, potato tubers transformed with synthetic LT-B gene have been reported higher LT-B concentration than bacteria-derived LT-B gene (Mason et al, 1998) and recently reported the possibility as a vaccine candidate of transgenic maize expressing ApxIIA gene using embryogenic calli co-cultivation transformation method (Kim et al, 2009). Like these, the expression of antigen-protein in plants could determine the suitability of the plants as a host and the availability of plant derived-antigen protein by injecting more visible.…”

“…2G-J). An efficient protocol of transformation in maize have been reported for immature embryo or embryogenic calli co-cultivation system using Agrobacterium (Ishida et al, 1996;Frame et al, 2002;Cho et al, 2005;Kim et al, 2009). According to many studies, the immature embryos of HiII genotype, that was produced type II callus, had been used for maize transformation (Frame et al, 2002).…”

“…Based on the number of independent resistant calli on selection medium, Frame et al (2002) and Utomo (2005) reported transformation efficiency of maize was 5.5 and 2.6%. Especially, Kim et al (2009) reported the transformation efficiency (0.6%) of maize was decreased when embryogenic calli of HiII genotype used as explant. In this study, based on the number of independent paromomycin-resistant calli recovered, the transformation efficiency of immature embryos was higher 2.1% than embryogenic calli (Kim et al, 2009).…”

“…Especially, Kim et al (2009) reported the transformation efficiency (0.6%) of maize was decreased when embryogenic calli of HiII genotype used as explant. In this study, based on the number of independent paromomycin-resistant calli recovered, the transformation efficiency of immature embryos was higher 2.1% than embryogenic calli (Kim et al, 2009). Sixteen T 0 putative transgenic plants have been produced as a result of 8 experiments.…”