Roses are known to be recalcitrant to in vitro regeneration methods. Regeneration via de novo shoot organogenesis or somatic embryogenesis is the key step for the successful process of genetic transformation and functional validation of genes. To improve the de novo shoot organogenesis process in rose genotypes, it is essential to control the physiological state of donor plants in order to prepare explant tissues and increase their regeneration abilities. Therefore, the aim of this study was to improve donor plant in vitro culture and mainly: (1) to understand the effect of media composition, including cytokinin and carbohydrate sources on cultured node morphogenesis, (2) to verify a possible interaction between genotype and media content and (3) to evaluate the best composition of the medium for a long in vitro storage to manage collections by increasing subculture period of four donor plant rose genotypes. All genotypes were able to increase their multiplication rate when they were subcultured on medium supplemented with benzylaminopurine, (BAP) 3 mg/L and high concentration of carbohydrate except sorbitol. Fructose or glucose was the best carbohydrate sources for multiplication but the optimal dose depends on genotype. The maximum of shoot elongation was obtained on medium including 0.5 mg/L benzylaminopurine and 0.1 mg/L gibberellic acid supplemented with 30 g/L fructose or glucose. Callus formation and rooting were enhanced by high concentration of fructose, glucose or sucrose. Maltose and sorbitol allowed an extended storage time for all genotypes. On the other hand, leaf explants issued from medium containing high concentration of benzylaminopurine and lacking gibberellic acid were able to regenerate adventitious shoot after transfer on shoot regeneration medium if combined with the optimal carbohydrate type and concentration which allow multiplication for the mother plants.