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.

Problem statement: Plant-based vaccines posses some advantages over other types of vaccine biotechnology such as safety, low cost of mass vaccination programs and wider use of vaccines for veterinary medicine. These study was undertaken to develop the transgenic maize as edible vaccine candidate for animals. Approach: The immature embryos of HiII genotype were inoculated with A. tumefaciens strain C58C1 containing the binary vector V622. The vector was harbored nptII gene, which confers resistance to paromomycin and ApxIIA gene was produced ApxII toxin, which was generated in various serum types of A. pleuropneumoniae as a target gene. Results: The 1,027 immature embryos were immersed for 5 min in the Agrobacterium solution and then these were co-cultured on solid cocultivation medium at 28°C for 2 days. After the delay period, the scutellum explants, axis removed embryos, were cultured on medium with 50 mg L −1 paromomycin for first 2 weeks and a paromomycinresistant callus were sorted out on the selection medium with 100 mg L −1 paromomycin for 4×14 days. A total of twenty callus clones were selected and sixteen-putative transgenic plants were regenerated. Among them, only five plants contained the integrated nptII gene, which was confirmed by Southern blot analysis. Conclusion: These results demonstrated that the nptII and ApxIIA genes integrated into the maize genome and that transgenic maizes can be use as vaccine candidate.