Protoplast Culture (type)

Having been isolated and fused (section 5.3.2.6), a protoplast still has to be cultured under carefully controlled conditions before it can regenerate into a plant. Culturing is carried out in MS liquid medium, with modifications such as droplet culture, plating, or micro-drop array techniques used to hold the fragile, wall-less protoplasts in place; before culturing begins, protoplast viability is checked using the dye fluorescein diacetate. Cultures are then incubated under continuous light of 1000-2000 lux at 25 degC - under these conditions, a new cell wall re-forms around each protoplast within 24-48 hours, and the first cell division typically occurs between 2 and 7 days into the culture. When two protoplasts from different parental cells have fused (section 5.3.2.6) but only their cytoplasm - not yet their nuclei - has combined, the resulting cell is called a cybrid; true nuclear fusion follows afterward, in a process called somatic hybridization. Because a mixed population of unfused, self-fused and cross-fused protoplasts always results from a fusion experiment, an essential final step is the selection of genuine somatic hybrid cells from among them. Figure 5.8 traces the …