Sixteen unvitrified shoots were produced through cell line technique on a recommended media (Emara et al. 2008). These media were, MS medium supplemented with 0.5 mg/l BA + 1.5 mg/l NAA for callus formation, and MS medium contained 1.0 mg/l BA for callus differentiation. The effect of number of subculture and the clone type on the growth and development of these clones was evaluated by subculturing the sixteen clones for five subcultures on MS medium supplemented with 2.0 mg/l BA. Subculture number 3 significantly showed the highest shoot number, leaf number and fresh weight/plantlet compared to all other subcultures. Concerning the effect of the clone type, clones number 9 and 10 observed the highest response in the same parameters mentioned above. The obtained plantlets of all clones were successfully acclimatized. However, during the acclimatization, plantlets of clones 11 and 10 showed the highest responses of survival percentage (95% and 92.33, respectively), and clones 9 and 10 observed high responses in the other growth parameters (shoot and leaf number/plantlet and plantlet height). Interestingly, the RAPD analysis of nuclear DNA confirmed that all the sixteen clones were different from the mother plant. Moreover, most of these clones showed some differences between each other. Accordingly, recommendation can be raised as to produce unvitrified plantlets as new clones of green nightshade (Solanum viride Solander ex Forst. f.) through cell line technique.