For callus induction, explants were excised from the roots of mulberry seedlings grown in a medium containing 10-5 M 6-benzylaminopurine and subject to culture in a solid medium supplemented with 10-6 M 2, 4-dichlorophenoxyacetic acid and 10-6 M thidiazuron. The isolated calli were repeatedly subcultured in the liquid medium, resulting in a faster-growing callus line. In this callus line, clumps of 20-30 cells, which were highly meristematic, were released into the medium. Protoplasts were enzymatically isolated from these clumped cells, and transfer of the B-glucuronidase (GUS) gene by electroporation was carried out at various pulse voltages. Histochemical observation showed that successful transient expression of the GUS gene was accomplished in 20-30% of protoplasts at the specified pulse voltages
