title: Stable chloroplast transformation of immature scutella and inflorescences in wheat (Triticum aestivum L.).
abstract: Chloroplast transformation in wheat was achieved by bombardment of scutella from immature embryos and immature inflorescences, respectively. A wheat chloroplast site-specific expression vector, pBAGNRK, was constructed by placing an expression cassette containing neomycin phosphotransferase 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 T(1) progeny seedlings. PCR analysis of gfp gene also confirmed the inheritance of transgene in the T(1) progeny. These 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.
authors.affiliation: China-UK HUST-RRes Genetic Engineering and Genomics Joint Laboratory, The Genetic Engineering International Cooperation Base of Ministry of Science and Technology, College of Life Science and Technology, Huazhong University of Science & Technology, Wuhan, China.
authors.forename: Cuiju
authors.initials: C
authors.lastname: Cui
authors.forename: Fei
authors.initials: F
authors.lastname: Song
authors.forename: Yi
authors.initials: Y
authors.lastname: Tan
authors.forename: Xuan
authors.initials: X
authors.lastname: Zhou
authors.forename: Wen
authors.initials: W
authors.lastname: Zhao
authors.forename: Fengyun
authors.initials: F
authors.lastname: Ma
authors.forename: Yunyi
authors.initials: Y
authors.lastname: Liu
authors.forename: Javeed
authors.initials: J
authors.lastname: Hussain
authors.forename: Yuesheng
authors.initials: Y
authors.lastname: Wang
authors.forename: Guangxiao
authors.initials: G
authors.lastname: Yang
authors.forename: Guangyuan
authors.initials: G
authors.lastname: He
mesh_terms: D015139:Blotting, Southern; D015153:Blotting, Western; D002736:Chloroplasts / Q000235:genetics* / Q000378:metabolism; D018742:DNA, Chloroplast / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D056626:Inflorescence / Q000235:genetics* / Q000378:metabolism; D019868:Kanamycin Kinase / Q000235:genetics; D018613:Microscopy, Confocal; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D018514:Plant Structures / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics; D014908:Triticum / Q000235:genetics* / Q000378:metabolism
chemical_list: D018742:DNA, Chloroplast; D049452:Green Fluorescent Proteins; D019868:Kanamycin Kinase