Transformation System of Rice Suspension-Cultured Microcolonies by Electroporation

Transformation System of Rice Suspension-Cultured Microcolonies by Electroporation

For establishing a transformation system of rice (Oryza sativa), after three days of culture embryogenic suspension-cultured cell clusters were enzymatically macerated for 2 hours in electroporation buffer containing 2% cellulase and filtered through 550, 400,250 and 100 ㎛stalnless mesh. Filtered embryogenic microcolonies of 100-250 ㎛with pB[12] were electroporated at 400 V/cm for 1.2 ms. Four weeks after the electroporation, stable transformed calli were obtained at a frequency of 72% on the selection medium containing 100 mg/L kanamycin. GUS gene in the genomic DNA among 20 out of 22 putative transformed calli lines were detected by PCR analysis. The expression of GUS gene into the kanamycin-resistance calli was confirmed by spectrophotometric assay and histochemical assay of GUS activity. In a histochemical study of the transgenic rice regenerants, it was shown that the GUS activity directed by the CaMV 35S promoter was localized mainly in leaf vein and root apex. Keywords: transformation system, embryogenic microcolonies, electroporation, Oryza sative, PCR, GUS expression