Budding and cleavage division of tobacco mesophyll protoplasts in relation to pseudo-wall and wall formation

Two saline media, differing primarily in the presence or absence of NH4 (+) but also in the concentration of sucrose, were developed for culture of tobacco (Nicotiana tabacum L.) mesophyll protoplasts. In the R0.6 medium, which does not contain NH4 (+) and only 1 g/l sucrose, protoplasts divide 2-3 times by budding and form only a pseudo-wall, i.e. a nonrigid structure containing polysaccharides. Later the cells degenerate, and sustained division does not take place. In the W 0.6 medium, which contains NH4 (+) and 30 g/l sucrose, the protoplasts form a rigid wall and divide by cleavage of the cells. After a few divisions, the walls of practically all of the newly formed cells degenerate into pseudo-walls, and the divisions cease. Only a few cells keep a wall, continue to divide, and form colonies. A very high frequency of colony formations from protoplasts is obtained by culturing protoplasts for a week in R0.6 or W 0.6 and then diluting the culture with a sugar medium. A detailled study of the inorganic and organic components of the saline media showed a strong interaction between the nitrogen supply and the cytokinin requirement. The advantages of the saline media in obtaining cell colonies from protoplasts, the problems associated with budding-type division, the causes of the cessation of division when no complete wall is formed, and the conditions necessary for wall formation are discussed.