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

• Published in: Plant cell reports Vol. 13; no. 3/4; pp. 188 - 192
• Main Authors: Golds, T.J, Babczinsky, J, Mordhourst, A.P, Koop, H.U
• Format: Journal Article
• Language: English
• Published: Germany 1994
• Subjects: alginate film culture technique; alginates; cell suspension culture; centrifugation; cultivars; culture techniques; Hordeum vulgare; mesophyll; Nicotiana tabacum; protoplasts; regenerative ability
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/BF00239890

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 x 30 min washing regime was found ti- 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 microliter) single cell aggregates were used to isolate and culture protoplasts.