Background: Calligonum polygonoides L. subsp. comosum (L'Hér.) Sosk. is a plant species belonging to family Polygonaceae. Susceptibility to threaten, presence of various chemical constituents, and many medicinal effects reported for this plant in addition to rareness of in vitro culture studies have fuelled the need for its micropropagation and phytochemical investigations of the produced cultures. Objectives: To employ in vitro culture technique for ex situ conservation of C. polygonoides, using the fruit as an explant; establish callus and cell suspension cultures from in vitro germinated plantlets; investigate the production of phenolics through callus, redifferentiated shoot, and cell suspension cultures; attempt to enhance cell capacity to accumulate phenolics using salicylic acid and yeast extract and provide a brief demonstration of biosynthetic pathway leading to phenolic production. Materials and Methods: Modified Murashige and Skoog media supplemented with growth hormones such as kinetin, 1-naphthaleneacetic acid, 6-benzylaminopurine, and indole-3-acetic acid were used to establish callus, redifferentiated shoots, and cell suspension cultures. Elicitation of cell suspension culture was performed using salicylic acid and yeast extracts. A reversed phase-high performance liquid chromatography method for determination of phenolic content in the aforementioned cultures was developed. Results: The unorganized callus and cell suspension cultures contained fewer amounts of phenolic compounds than redifferentiated shoots. Elicitation produced massive quantitative reprogramming of phenolic content. Conclusion: The present study offers an alternative and renewable source for this valuable natural plant, provide a chance to improve secondary metabolite yield and serve as a useful tool for studying the biosynthesis of these compounds and its regulation in plant cells.