In vitro shoot regeneration from leaf mesophyll protoplasts of hybrid poplar (Populus nigra x P. maximowiczii)

Protoplasts were isolated from leaf mesophyll of hybrid poplar (Populus nigra X P. maximowiczii) with a mean yield of 10.4 x 10(6) protoplasts per g fresh weight using 2.0% Cellulase 'Onozuka' R-10, 0.8% Macerozyme R-10, 1.2% Hemicellulase, 2.0% Driselase, and 0.05% Pectolyase Y-23 with CPW salts solution containing 0.6 M mannitol, 0.002 M DTT, 3 mM MES at pH 5.6. A liquid plating method produced the highest frequency of dividing protoplasts (48.6%) using an MS medium without NH4NO3. The highest percent of colony formation was 22.8%, produced with fabric supported semi-solid (0.5% w/v) agar plating method using the same culture medium. Growing cell colonies and/or micro-calli were transferred to a fresh semisolid agar medium containing 0.44 μM BAP and 9.0 μM 2,4-D. Multiple shoots were produced from protoplast-derived callus after culture on MS medium containing 6.8 μM zeatin. After root induction on half-strength MS medium that lacked growth regulators, shoots were transferred to pots containing artificial soil mix.