Nonenzymatic Release of Intact Protoplasts from Mature Pollen of Bean

Abstract

A nonenzymatic technique using dilute salt solutions effected rapid release of viable protoplasts from mature bean (Phaseolus vulgaris L.) pollen. Protoplasm release started within 30 sec and was completed within 5 min in solutions of 0.02 to 0.06 m NaCl, or KCl, pH 6 to 9. The degree of release could be altered by changing the concentrations and ratios of CaCl2 and H3BO3 and by adding sucrose to either solidified or aqueous salt media. The surface of nonenzymatically released protoplasts was partially digested by short-time exposure to a mixture of cell wall-degrading enzymes and then examined by scanning electron microscope.