CHLOROPLAST transformation in wheat was achieved by ……..bombardment of callus from immature embryos. A wheat chloroplast site-specific expression vector pMY-Wt-nif was constructed by placing an expression cassette containing neomycin phosphotransferase II (nptII) and nitrogen fixation gene cluster from Clostridium acetobutylicum in the intergenic spacer between trnI and trnA of the wheat chloroplast genome. Integration of nitrogen fixation gene cluster in the plastome was identified by polymerase chain reaction (PCR) analysis and Southern blotting using a specific sequence of the cassette as a probe. Expression of nitrogen fixation gene cluster was examined by Western blot. The results strengthen the feasibility of wheat chloroplast transformation and also give a novel method for the introduction of important agronomic traits in wheat through chloroplast transformation.
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