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Tsutomu Shimizu
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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.
Co-Authors I Putu Suparthana Masahiro Nogawa Mineo Kojima