Abstract

We report an efficient procedure for transformation of young leaf disc explants ofPerilla frutescens using Agrobacterium tumefaciens strain 4404 harboring the binary vector pYB1130, which contains T-DNA incorporating the g-TMT gene driven by the CaMV 35S promoter and the neomycin-phosphotransferase (npt II) gene for kanamycin selection. Explants were cultured on modified MS medium supplemented with 0.1 mg/l TDZ, 30 g/l sucrose, 100 mg/l kanamycin and 250 mg/l cefatoxime. Transformed shoots emerged from the basal part of the leaf after 4 to 5 weeks and had normal phenotypes when grown in the field. To improve transformation efficiency, the following factors were examined; plant growth regulators, kanamycin sensitivity, inoculation time and co-cultivation period. The highest number of transformants was obtained when leaf explants were co-cultivated for 3 days with Agrobacterium in the dark at 24°C. The optimum time of inoculation was 7 min, resulting in 17 transgenic plants. Stable integration of T-DNA into the genome of the putative transgenic plants was confirmed by polymerase chain reaction amplification (PCR) and Northern blot analysis. This method can be efficiently used for stable transformation of P. frutescens in which the α-tocopherol content was enhanced by the overexpression of this gene.

Key words: Co-cultivation, overexpression, Perilla frutescens, g-TMT. α-tocopherol

Abbreviation

CTAB,  Cetyltrimethylammonium bromide; HPLC, high performance liquid chromatography; MS medium,  Murashige-Skoog medium; NPT II, neomycin phosphotransferase gene; TDZ, thidiazuron; g-TMT, g-tocopherol methyltransferase.