Large Electro-Fused Protoplasts of Populus albaSelected by a Micromanipulator: Techniques and Some Characteristics of Cells and Their Regenerants

An improved method for selection of large electro-fused protoplasts ofPopulus albaby a micromanipulator was developed. The conditions for electric cell fusion treatment were optimized. For the best result, protoplasts with a cell density of 5 × 105/mL were treated with an alternate current (1 MHz, 200 V/cm) and pulsed with a direct current (2 kV/cm) for 100μs in 2.5 mM CaCl2and 0.55 M mannitol. The electo-fused protoplasts were cultured in NH4NO3-free Murashige and Skoog's medium containing 0.6 M of mannitol, 0.09 M sucrose, 1μM of 2,4-dichlorophenoxyacetic acid and 0.1μM of benzyladenine, the same medium used for protoplast culture, but at a very low cell density of 5–10 × 102/mL in a well of a 96-well culture plate. When cell aggregates derived from individual fused protoplasts were transferred to fresh medium with 0, 0.3 or 0.6 M mannitol, large colonies developed. In the shoot differentiation medium, the reaction of the calluses derived from large fused protoplasts towards the growth regulators differed from the non-fused ones. In medium containing 1μM each of naphthalene acetic acid andN-(2-chloro-4-pyridyl)-N′-phenylurea, growth of callus from electro-fused ones was not reduced by much compared to the control, but shoot differentiation was inhibited. Gibberellic acid (0.1–10μM) was beneficial to shoot regeneration; however, irregularly shaped leaves appeared at high gibberellic acid concentrations. Shoots regenerated were rooted in Murashige and Skoog's medium containing 4μM of indole-3-butyric acid. Some plantlets obtained had a varied morphology. Based on the characteristics of growth, some cells derived from electro-fused protoplasts appear to be physiologically different from the non-fused one.