Abstract
Selection of glyphosate-resistant clones from MNNG-treated mesophyll protoplasts of haploid tobacco and their differentiation were studied. The protoplasts were treated with 0.1 to 100 $\mu\textrm{g}$/mL N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for 30 min when they expanded to oval shapes. After the treatment, the protoplasts in 4-16 cell stages were transferred to the selective medium containing 1 mM glyphosate for the selection of the glyphosate-resistant colonies. The efficiency of the cell division of the protoplasts in the selective medium decreased as the MNNG concentrations in creased. Optimal MNNG concentration for induction of the glyphosate-resistant clones was 10$\mu\textrm{g}$/mL and mutation frequency was 2.66$\times$10-6. The stability of the glypohsate-resistance of the clones was examined by prolonged subculture in the medium with 1 mM glyphosate, and the resistant clones were survived more than 10 months. Among them one clone has been proliferating and greening and the others were proliferating without greening or greening with slower proliferating.