Abstract: Transgenic technology plays an important role in gene function analysis and crop germplasm innovation. However, transgenic studies are relatively recent in chicory. In this study we sought to improve tissue culture and genetic transformation methodology in chicory. Cotyledons grown in the dark were collected as explants. After transformation mediated by Agrobacteria, these explants were grown under illuminated conditions. The cotyledon cells divided rapidly when they were transferred from dark to light. The emergence rate of regenerated seedlings was greater than 90%, similar to that of previous studies however, the germination rate could be improved by 80%-90% if the explants were not pre-cultured. Our results were time, money and labor-saving. We also found that during callus induction, plant differentiation and root growth, decreased intensity and concentration of cytokinin significantly reduced the vitrification of callus and regenerated plantlets. Compared with continuously applied cytokinin our method decreased vitrification of callus and regenerated plantlets by about 9% and 16% respectively.