The objective of this research was in-vitro germination and callus induction of Onosma bulbotrichum ( O. bulbotrichum) as a medicinal herb which belongs to Boraginaceae family. For germination, the seeds were cultured on growth regulator-free MS medium and for callus induction, seeds were sown on modified MS medium containing different concentrations of kinetin (kn)- Indole-3-acetic acid (IAA) and kn- 2,4-D (2,4-dichlorophenoxyacetic acid), respectively. The plates were maintained in the dark at growth chamber. After 7 days seed germination on hormone-free medium and after 10 days callus initiation on modified medium in the presence of hormones was occurred. The maximum pigmented callus (100%) was observed on modified MS medium with a combination of 0.2 mg.L -1 IAA + 2.10 mg.L -1 kn. Shikonin determination was performed by HPLC method. In addition, total hydroxynaphtoquinons as polyphenols in sum of callus and culture medium were measured by spectrophotometric method and revealed that total naphtoquinones content at IAA was more than 2, 4-D.