Isolation and Regeneration of Tobacco Mesophyll Cell Protoplasts under Low Osmotic Conditions

Abstract

A method is described for the isolation of large numbers of tobacco (Nicotiana tabacum L. cv. Xanthi-nc) mesophyll cell protoplasts under relatively low external osmotic conditions. The procedure utilized 0.2 m sucrose as the primary osmoticum and a mixture of 0.5% macerozyme, 4% cellulase, and 2% polyvinylpyrrolidone, pH 5.4. The viability of resultant protoplasts was confirmed through regeneration of fertile plants. Plating and regeneration studies revealed, however, that qualitative and quantitative modifications in plating and differentiation media were necessary for protoplasts prepared in this manner. Over-all, the procedure was found to be a simplified alternative to those previously described for tobacco protoplast regeneration. In addition, the system should permit studies related to the influence of differing osmoticum levels on a variety of cell functions.