Isolation and properties of protoplasts from endosperm of developing wheat grain

Abstract

Conditions have been systematically evaluated for the isolation of viable protoplasts from endosperm tissue of developing wheat grain ( Triticum aestivum L. cv. Mardler). Optimal conditions are as follows: an enzyme-mixture of 1% pectinase and 2% cellulase in 0.3 M mannitol and 0.2 M sucrose as osmoticum at pH 5.5; 2 h at 28°C or 16 h at 4°C incubation; 1 × g sedimentation for protoplast purification; centrifugation and abrupt changes in pH or temperature should be avoided to prevent protoplast lysis. A yield of 1–5 × 10 5 endosperm protoplasts per gram fresh weight of grain was obtained with this procedure. This was estimated to represent 10–50% of the original endosperm cells. Approximately 80% of the protoplasts excluded Evans blue dye. Enzyme latency experiments measuring uridine diphosphate glucose (UDPG) pyrophosphorylase activity showed that more than 80% of the protoplasts were intact. The protoplasts were able to incorporate radiolabelled sucrose or glucose into starch at rates comparable to isolated endosperm tissues. These data indicate that the protoplasts are metabolically active and sufficiently free from contamination to permit further metabolic studies and the development of plastid isolation procedures.