Expression and genomic integration of transgenes after Agrobacterium-mediated transformation of mature barley embryos.
Mature embryos in tissue cultures are advantageous because of their abundance and rapid germination, which reduces genomic instability problems. In this study, 2-day-old isolated mature barley embryos were infected with 2 Agrobacterium hypervirulent strains (AGL1 and EHA105), followed by a 3-day period of co-cultivation in the presence of L-cystein amino acid. Chimeric expression of the b-glucuronidase gene (gusA) directed by a viral promoter of strawberry vein banding virus was observed in coleoptile epidermal cells and seminal roots in 5-day-old germinated seedlings. In addition to varying infectivity patterns in different strains, there was a higher ratio of transient b-glucuronidase expression in developing coleoptiles than in embryonic roots, indicating the high competency of shoot apical meristem cells in the mature embryo. A total of 548 explants were transformed and 156 plants developed to maturity on G418 media after 18-25 days. We detected transgenes in 74% of the screened plant leaves by polymerase chain reaction, and 49% of these expressed neomycin phosphotransferase II gene following AGL1 transformation. Ten randomly selected T0 transformants were analyzed using thermal asymmetric interlaced polymerase chain reaction and 24 fragments ranged between 200-600 base pairs were sequenced. Three of the sequences flanked with transferred-DNA showed high similarity to coding regions of the barley genome, including alpha tubulin5, homeobox 1, and mitochondrial 16S genes. We observed 70-200-base pair filler sequences only in the coding regions of barley in this study.
Department of Molecular Biology and Genetics, Faculty of Science, Istanbul University, Vezneciler, Istanbul, Turkey ucarlicu@istanbul.edu.tr.
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Uçarlı
Department of Molecular Biology and Genetics, Faculty of Science, Istanbul University, Vezneciler, Istanbul, Turkey.
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Tufan
Department of Molecular Biology and Genetics, Faculty of Science, Istanbul University, Vezneciler, Istanbul, Turkey.
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Gürel
D016960:Agrobacterium tumefaciens / Q000235:genetics; D005822:Genetic Vectors; D018745:Genome, Plant; D023281:Genomics; D018525:Germination / Q000235:genetics; D001467:Hordeum / Q000196:embryology / Q000235:genetics* / Q000254:growth & development; D019868:Kanamycin Kinase / Q000235:genetics; D018519:Meristem / Q000196:embryology / Q000235:genetics / Q000254:growth & development; D030821:Plants, Genetically Modified / Q000196:embryology / Q000235:genetics* / Q000254:growth & development; D036226:Seedlings / Q000196:embryology / Q000235:genetics* / Q000254:growth & development; D012639:Seeds / Q000235:genetics / Q000254:growth & development; D014170:Transformation, Genetic; D019076:Transgenes
D019868:Kanamycin Kinase