Requirement of thidiazuron for healthy protoplast development to efficient tree regeneration of a hybrid poplar ( Populus tremula ß p. alba)

Methods were developed for obtaining high yields of leaf protoplasts of a hybrid clone of white poplar, Populus tremula × P. alba (717-1-B4), as well as rapid and efficient plant regeneration. Protoplasts were isolated from leaves of shoot culture. A precise adaptation of liquid media for preparation and culture of protoplasts led to high rates of cell colony formation, on the average for 30% of initially plated protoplasts. The procedures employed avoided the formation of any exudate. Glucose (9%) as an osmoticum and carbon source was the only sugar leading to high viability. A precise balance of low concentrations of nitrate and ammonium in the culture medium was beneficial for development of healthy cell colonies. A combination of thidiazuron (0.05 µM) as the only cytokinin and 2,4-D (14 µM) as auxin was essential for sustained development of protoplast derived cell colonies. After dilution in liquid medium, about 10% of the colonies formed calli. Plants regenerated through bud formation on practically all protoplast-derived calli. Plants regenerated from 3% of the initially plated protoplasts. In vitro rooting and transfer to soil were also achieved with good efficiency.