Stable chloroplast transformation of immature scutella and inflorescences in wheat （Triticum aestivum L.）

Chloroplast transformation in wheat was achieved by bombardment of scutella from immature embryos and immature infiorescences, respectively. A wheat chloroplast sitespecific expression vector, pBAGNRK, was constructed by placing an expression cassette containing neomycin phos- photransferase II （nptII） and green fluorescent protein （gfp） as selection and reporter genes, respectively, in the intergenic spacer between atpB and rbcL of wheat chloroplast genome. Integration of gfp gene in the plastome was identified by polymerase chain reaction （PCR） analysis and Southern blotting using gfp gene as a probe. Expression of GFP protein was examined by western blot. Three positive transformants were obtained and the Southern blot of partial fragment of atpB and rbcL （targeting site） probes verified that one of them was homoplasmic. Stable expression of GFP fluorescence was confirmed by confocal microscopy in the leaf tissues from T1 progeny seedlings. PCR analysis of g~ gene also confirmed the inheritance of transgene in the T1 progeny. These results strengthen the feasibility of wheat chloroplast transformation and also give a novel method for the introduction of important agro- nomic traits in wheat through chloroplast transformation.