The induction of sensitivity to gibberellin in aleurone tissue of developing wheat grains : III. Sensitisation of isolated protoplasts.

A method for isolating viable protoplasts in high yield from the aleurone layers of developing wheat grains is described, and the techniques for their subsequent culture outlined. Protoplasts from untreated tissue do not produce α-amylase in response to gibberellic acid (GA3) if the incubation temperature is left at 25°C. However, pre-treatment of the protoplast preparation at temperatures above 27°C for at least 8 h followed by a short incubation at 25°C induces sensitivity to the growth regulator such that α-amylase is produced. The requirements of the sensitisation process are similar to those for intact aleurone tissue although additional adjustment to the calcium ion is beneficial. Pre-treatment of aleurone layers with the sensitising temperature regimes prior to protoplast isolation have the advantage of increasing protoplast viability. Once sensitised, the protoplasts respond to a GA3 concentration as low as 10(-11) mol dm(-3) with a maximal response at 10(-9) mol dm(-3). The successful isolation of wheat aleurone protoplasts whose sensitivity to GA3 can be manipulated represents a useful step towards investigating the role of cell membranes in growth-regulator action.