Plantlet regeneration from mesophyll protoplasts of Betula platyphylla var Japonica

In Betula platyphylla var. japonica, colonies were induced efficiently from mesophyll protoplasts cultured in half strength MS (1/2MS) liquid medium containing 0.6 M mannitol, 0.09 M sucrose and 1 mu M 4-PU and 1 mu M NAA at a cell density of 5 x 10(4)/ml. The colonies grew actively and developed into callus after 3 months of culture. Roots differentiated from the protoplast-derived white calluses cultured on the 1/2MS solid media supplemented with 0.1-1 mu M 4-PU and 1 mu M NAA, and 10 mu M zeatin with no supplementation of NAA. Furthermore, the protoplast-derived green callus differentiated shoots with 1/2MS solid medium containing 1 mu M 4-PU or 10 mu M zeatin with no supplementation of NAA. When shoots obtained were cultured on the cytokinin-free MS solid medium with 2.5 mu M IBA and 0.1 mu M NAA, they rooted and developed into plantlets after one month of culture. The phenylurea-type cytokinin, 4-PU, was effective for plantlet regeneration from the mesophyll protoplasts of B. platyphylla var. japonica. This suggests that there is potential for the use of 4-PU in the culture of protoplasts in many forest tree species.