Electroporation에 의한 벼 현탁배양 세포의 형질전환

Transformation of Rice Suspension-Cultures by Electroporation

Calli were induced from the leaf base region of germinated rice (Oryza sativa L. cv Nakdong) on LS medium supplemented with 2.5mg/L 2,4-D in the dark at 27℃. Embryogenic calli of pale yellow and globular type were selected and used for the initiation of cell suspension cultures is AA₂ liquid medium with 2mg/L 2,4-D, 0.2mg/L kinetin and 0.1mg/L GA₃ For rice transformation, suspension-cultured cells were enzymatically macerated for 2 hours in 2% Cellulase solution and filtered through 520, 380, 230 and 90㎛ stainless mesh. Filtered embryogenic microcolonies of 90-230/㎛ with pBl121 were electroporated at 400 V/cm for 1 ms. Transformed calli were selected from the electroporated calli on 100mg/L kanamycin-containing media. GUS gene in the genomic DNA of 5 putative transformed callus lines were detected by PCR. The expression of GUS gene in the kanamycin-resistance calli was confirmed by spectrophotometric assay and histochemical assay of GUS activity. Regenerants from the kanamycin-resistance calli showed high specific expression of GUS gene.