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Agrobacterium-mediated transformation technology for monocot Agrobacterium Monocot plant Callus (dedifferentiated tissue) Scutellum of Immature embryo rice calli immature embryo Copyrights © 2009 Japan Tobacco Inc. All rights reserved. maize immature embryo 1 Monocot transformation technology Target plants Corn Rice Wheat Barley Switchgrass Miscanthus Sorghum Big bluestem Copyrights © 2009 Japan Tobacco Inc. All rights reserved. Rye Oat Millet Turf Banana Pineapple Sugarcane 2 Agrobacterium Common soil bacteria DNA transfer capability Wild-type Agrobacterium makes crown gall via its gene transfer Copyrights © 2009 Japan Tobacco Inc. All rights reserved. 3 T-DNA transfer mechanism Signal Molecule Induction of Vir Gene Expression P P Activation Vir Region Expression Integration Ti Plasmid RB LB Assemble T-DNA Generation of ss T-strand Maturation of T-complex T-complex Agrobacterium tumefaciens Copyrights © 2009 Japan Tobacco Inc. All rights reserved. T-complex Channel Complex Targeting to Nucleus Plant Cell 4 Super-binary system, more efficient S Vir T-DNA Hybrid vector Vir region T-DNA ORI Binary System ori cos S Vir r oriSp Tetr cos Homologous Recombination S Vir region T-DNA Vir region Acceptor Vector ori cos Tetr ORI ori cos Super-binary System Spr Intermediate Vector T-DNA Copyrights © 2009 Japan Tobacco Inc. All rights reserved. 5 Vectors and Strains Transient assay using rice calli LBA4404 pBI EHA101 LBA4404 pBI pSB Tsukinohikari Koshihikari Copyrights © 2009 Japan Tobacco Inc. All rights reserved. 6 Strengths Able to introduce DNA with defined ends. Able to introduce relatively large fragment. Low copy. Low frequency of gene silencing. Low frequency of somaclonal variations. High fertility and high quality. Industrial standard Copyrights © 2009 Japan Tobacco Inc. All rights reserved. 7 Publications Articles Hiei Y., Ohta S., Komari T. and Kumashiro T. Ishida Y, Saito H, Ohta S, Hiei Y, Komari T and Kumashiro T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6: 271 (1994) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nature Biotech. 14: 745 (1996) Hiei, Y., and Komari, T. Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens. Plant Cell, Tissue and Organ Culture 85, 271-283 (2006) Copyrights © 2009 Japan Tobacco Inc. All rights reserved. 8 Publications Patents Callus (dedifferentiated and dedifferentiating tissues) Method of transforming monocotyledon WO 94/00977 Immature embryo Method of transforming monocotyledon by using scutellum of immature embryo. WO 95/06722 Copyrights © 2009 Japan Tobacco Inc. All rights reserved. 9