Efficient plant regeneration from protoplasts isolated from long-term, shoot primordia-derived calluses of garlic (Allium sativum)

We developed an efficient method for the regeneration of plants from garlic (Allium sativum) protoplasts. Protoplasts were isolated from six-year-old cultures of shoot primordia-derived calluses. At the isolation condition applied, high yield of protoplasts was obtained from the calluses compared with that from the initial shoot primordia. Throughout long-term culturing, highly regenerative calluses that propagated rapidly and appeared genetically stable were selected. The frequency of colony formation from protoplasts was clearly enhanced by increasing cell density. Adventitious buds were induced from protoplast-derived calluses at ca. 90% efficiency, and the regeneration efficiency from adventitious buds averaged nearly 70%. Plants that regenerated from adventitious shoots showed normal phenotypes, suggesting that long-term culturing of these garlic calluses does not induce dramatic genetic alterations.