Isolation of protoplasts from stem and hypocotyl of the legumeVigna sinensis and some factors affecting their regeneration

Summary

Cut segments of stem and hypocotyl ofin vitro grown seedlings were incubated in 1% cellulase, 0.6 M mannitol and a salt solution at pH 5.5 for 10 hours. When these segments were left undisturbed the protoplasts formed did not escape into the enzyme solution which was removed by decantation. Also the tissue could be washed and liquid removed by decantation. Finally the protoplasts were released by shreding the tissue followed by gentle shaking. Undigested material was removed by sieving. The preparation thus obtained hardly had any cell requiring purification. In this way protoplasts could be obtained without centrifugation—a necessary evil in current techniques of harvesting of protoplast. These protoplasts readily regenerated if isolated from plants grown at 3,500 lux. The growth, however, did not proceed beyond a few-celled stage unless the pH was adjusted to 6.0. For sustained divisions osmotic concentration of the nutrient medium also had to be lowered to 0.5 M. This method, however, could not be applied to mesophyll protoplasts which required centrifugation for washing and purification resulting in heavy losses in yield and destabilization of protoplasts which failed to divide.