Micropropagation, genetic engineering, and molecular biology of Populus

Protoplasts are cells without cell walls. In plants protoplasts are usually liberated by tissue digestion with cellulases and pectinases purified from wood-rotting fungi. To keep the plasma membrane from bursting during protoplast isolation, an isotonic or slightly hypertonic medium is used. Because of the lack of external constraints on the membrane, protoplasts are spherical (figure 1B). One use of plant protoplasts is to isolate single cells for physiological studies. In Populus (poplar), protoplasts have been used to study reactions of the photosynthetic pathway (Dalakishvili et al. 1989; Mgaloblishvili et al. 1988; Sanadze et al. 1986, 1990). Mter the demonstration of totipotency of tobacco protoplasts by Takebe et al. (1971), many laboratories began developing protocols to regenerate plants from herbaceous and woody plant protoplasts to use in genetic engineering. Protoplasts were considered essential for gene transfer because the cell wall is usually impermeable to DNA. Several protoplast-based genetic engineering techniques emerged, including protoplast fusion, DNA microinjection, electroporation, and polyethylene glycol (PEG)-mediated gene transfer. Today, other methods, such as Agrobacteriummediated gene transfer and biolistics, are available to transform cells within intact tissues. Still, protoplasts are 1 method that has proven valuable for genetic engineering research. To obtain transgenic plants, the genetically altered protoplasts must regrow their cell walls, divide, and regener-