2001 Volume 70 Issue 4 Pages 431-437
For the purpose of establishing protoplast culture systems in Japanese bunching onion (Allium fistulosum L.), based on BDS inorganic salts (Dunstan and Short, 1977), nutrient media suitable for protoplast culture were investigated. The optimum cell division was achieved with 5 mM potassium nitrate and a combination of 2μM 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.2 or 1 μM 6-benzylaminopurine (BAP). The suitable osmolality for protoplast culture was around 0.60 osmol·kg-1, whereas a combination of 0.2 M sucrose and 0.2 M glucose accelerated cell division. The protoplasts developed into colonies by gradual reduction of sugar concentration. After 45 days of culture, numerous micro-calli formed which were subsequently transferred to a callus formation medium. Plantlets were regenerated by transferring protoplast-derived calli of ca. 2 mm in diameter to a modified Murashige-Skoog medium. Of the 75 calli inoculated, 24 (32%) gave rise to green shoots on a regeneration medium 2 to 3 months after inoculation. These shoots rooted when cultured on a hormone-free medium. These plantlets were successfully acclimatized and grew normally in a greenhouse.