Protoplast preparation without centrifugation: plant regeneration of barley (Hordeum vulgare L.).

Using a modification of the alginate film culture technique we show that it is possible to prepare and culture tobacco mesophyll and barley cell suspension protoplasts without centrifugation. Comparable division frequencies and colony development were observed from protoplasts embedded with enzyme and protoplasts purified by centrifugation. A 3 × 30 min washing regime was found to be the minimum time necessary to remove the enzyme from the gelled alginate matrix. The procedure provides a more gentle method for isolating protoplasts. It has the additional benefit of recovering all of the cells released from the starting tissue. In particular, the smaller protoplasts that are frequently lost during conventional isolation, are maintained. In barley, we illustrate the use of the system for recovering plants from embryogenic protoplast-derived calli from the cultivars Dissa and Igri. Finally, using small volumes of enzyme (50 μl) single cell aggregates were used to isolate and culture protoplasts.