A SIMPLE AND EFFICIENT GENE-TRANSFER SYSTEM OF TRIFOLIATE ORANGE (PONCIRUS-TRIFOLIATA RAF)

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 pBI101-012-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 mu g/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.