INFLUENCE OF ELECTRICAL TREATMENT AND CELL-FUSION ON CELL-PROLIFERATION CAPACITY OF SUNFLOWER PROTOPLASTS IN VERY-LOW-DENSITY CULTURE

Sunflower hypocotyl protoplasts were cultivated as either single cell or in a very low cell density culture, in a completely defined medium without feeder cells. The cells were cultivated at a density of only 4 -20 protoplasts/ml which is some three orders of magnitude smaller tha n the usual cultures for sunflower protoplasts. In our cultures develo pment of colonies up to a microcallus stage occurred within 40 days. T he frequency of microcallus formation from protoplasts increased drama tically when the protoplasts, prior to cultivation, were subjected to short exposures to AC electric fields from less than 1% without pre-cu lture electrical treatment to greater than 30% with treatment. Protopl asts assembled in single pairs were also fused using pulsed electric f ields. Electrical fusion between individual pairs of cells was achieve d in 30% of attempts. More than 70% of these fusion products proceeded to divide. The results obtained have important ramifications since si ngle cell and low density cell culture offers considerable advantages for hybrid production and bio-engineering of plants such as sunflower. (C) 1997 Elsevier Science Ireland Ltd.