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Mineo Kojima
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Biochemistry, Genetics & Molecular Biology Environmental Science

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ANALYSIS OF GENE TARGETING EVENTS IN BUCKWHEAT (Fagopyrum esculentum) TRANSFORMED b THE MUTANT STRAINS OF Agrobacterium tumefaciens THAT HAVE DEFICIENCY IN T-­??DNA INTEGRATION STEP INTO PLANT GENOME Mineo Kojima; Putu Suparthana; Tsutomu Shimizu; Masahiro Nogawa
International Journal of Biosciences and Biotechnology Vol 2 No 1 (2014)
Publisher : Central Laboratory for Genetic Resource and Molecular Biology, Faculty of Agriculture, Udayana University in cooperation with Asia-Oceania Bioscience and Biotechnology Consortium (AOBBC)

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Abstract

Gene targeting events were analyzed in buckwheat (Fagopyrum esculentum, var. Shinano No. 1) that werein planta transformed (T1) by the three strains of Agrobacterium tumefaciens harboring a gene targetingvector for an endogenous gene (acc.no.AB327276); M-­??1 mutant (abvA::Tn5) and M-­??31 mutant (virA::Tn5)strains, both of which are capable of transferring its T-­??DNA into nucleus of host plant cell, but have adeficiency in T-­??DNA integration step into chromosome and LBA4404 strain, being a commonly usedstrain. The results of both phenotype examination and Southern blot analysis implied that a targetingconstruct(s) was integrated into a different locus (loci) each depending on the strain of A. tumefaciens usedfor transformation. Both 5’-­??end and 3’-­??end flanking DNA segments that were expected from preciseinsertion of targeting construct into the endogenous U-­??gene locus were obtained by PCR with 3 (23%)transformants out of the randomly selected 13 transformants by M-­??1 mutant strain, while only 3’-­??endflanking DNA segment was obtained with 3(21%) transformants out of 14 transformants by M-­??31 strain.Taken together, the results suggested the potential of usage of a mutant strain of A. tumefaciens for genetargeting which has a deficiency in T-­??DNA integration step into plant genome.
DEVELOPMENT OF IN PLANTA TRANSFORMATION METHOD USING Agrobacterium tumefaciens THAT IS SIMPLE AND EFFICIENT AS WELL AS APPLICABLE TO VARIOUS PLANTS Mineo Kojima
International Journal of Biosciences and Biotechnology Vol 1 No 2 (2013)
Publisher : Central Laboratory for Genetic Resource and Molecular Biology, Faculty of Agriculture, Udayana University in cooperation with Asia-Oceania Bioscience and Biotechnology Consortium (AOBBC)

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Abstract

A simple and effi cient in planta transformation method was developed. In the method, meristems of eitherapical or axillary buds of immature plants or apical buds of embryos in imbibed seeds, depending on plants,were inoculated by Agrobacterium tumefaciens after being pricked with a needle. The inoculated plants weregrown to maturation in pots under non-sterile conditions. Transformation was demonstrated by severallines of evidence obtained with mostly the progenies of T1 generation; phenotypic inheritance from T0plants to plants of the following generation, resistance of seed germination to antibiotics, detection of?-glucuronidase activity in transformants of T1 generation, detection of transgene by Southern blot andPCR analyses in T1 generation transformants and rescue from T1 generation transformants of the plasmidscomposed of T-DNA of binary vector and fl anking plant genomic DNA. The diverse species of plants such asbuckwheat (Fagopyrum esculentum), mulberry (Morus alba L.), kenaf (Hibiscus cannabinus), rice (Oryza sativa),wheat (Triticum aestibvum L.), maize (Zea mays), and soybean (Glycine max) were shown to be effi cientlytransformed by our in planta method.
Co-Authors I Putu Suparthana Masahiro Nogawa Tsutomu Shimizu