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A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliata Raf.)
Authors:J Kaneyoshi  S Kobayashi  Y Nakamura  N Shigemoto  Y Doi
Institution:(1) Institute of Biotechnology, Hiroshima Prefectural Agricultural Research Center, Hachihonmatsu, 739-01 Higashi-Hiroshima, Japan;(2) Akitsu Branch, Fruit Tree Research Station, Akitsu, 729-24 Hiroshima, Japan
Abstract:Summary A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliata Raf.) was developed using epicotyl segments. The segments were infected with Agrobacterium harboring the binary vector pBI121 or pBI101-O12-p1. Both vectors contained the neomycin phosphotransferase II (NPTII) and the beta-glucuronidase (GUS) genes. In the plasmid pBI101-O12-p1, the GUS gene was directed to the promoter region of ORF12 (rolC) of the Ri plasmid. On a selection medium containing 100 or 200 mgrg/ml kanamycin, adventitious shoots were formed from 21.7–44.6% of the segments. Histochemical GUS assay showed that 55.4–87.7% of the shoots expressed the GUS gene. The stable integration of this gene was also confirmed by polymerase chain reaction (PCR) analysis and by Southern blot analysis. When the pBI101-O12-p1 plasmid was used, the GUS activity was found to be located in phloem cells of leaf, stem and root. More than 100 transformed plants were obtained using this method within 2–3 months.
Keywords:Trifoliate orange  Epicotyl segment  Agrobacterium  Transformation  rolC promoter  beta-glucuronidase" target="_blank">gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase
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